MXPA00009177A - Limanthes - Google Patents

Abstract

This invention relates to an isolated nucleic acid fragment encoding an enzyme involved in lipid biosynthesis. The invention also relates to the construction of a chimeric gene encoding all or a portion of the enzyme involved in lipid biosynthesis, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the enzyme involved in lipid biosynthesis in a transformed host cell.

Description

GENES FOR LIMNANTHES OIL FIELD OF THE INVENTION This invention relates to the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments that encode enzymes involved in the lipid biosynthesis in plants and seeds.

BACKGROUND OF THE INVENTION Improved means for handling fatty acid compositions, from biosynthetic or natural plant sources, is of paramount importance. For example, sources of edible oil containing minimal possible amounts of saturated fatty acids are desired for nutritional reasons and alternatives are needed to generate sources of highly saturated oil products, such as tropical oils. Fatty acids are used in plant membranes and in neutral lipids that are formed for energy storage in developing seed tissues. The composition of fatty acid (polarity, chain length and degree of unsaturation) of a membrane determines its physical REF: 121959 properties. The most common fatty acids contain 16 to 18 carbons (C16 or C18) with one or more double bonds. Fatty acids with larger (C20 or C22) or shorter (C12 or C14) carbon chains are uncommon compared to hydroxylated fatty acids and fatty acids with different positions of the double bonds (delta-5 or delta- 6). Higher plants appear to synthesize common fatty acids via a metabolic pathway in the plastid organelles of the plant (i.e., chloroplasts, proplástidos or other related organelles) with intermediaries attached to acyl carrier proteins as part of the fatty acid synthesis complex (FAS). The pathways involved in the synthesis of common fatty acids in developing oilseeds are not well understood and relatively easy to manipulate. In fatty acid biosynthesis, delta-9-acyl-desaturated lipid / delta-9-acyl-CoA desaturase most commonly introduces a double bond at the delta-9 position of a C18 saturated fatty acid (ie, the desaturation of stearoyl-ACP (C18: 0-ACP) to oleoyl-ACP (C18: 1-ACP)) to produce monounsaturated fatty acids. Various other fatty acid desaturases enzymes are known in higher plants such as delta-6 and delta-5 desaturases which further desaturate monounsaturated fatty acids to make polyunsaturated fatty acids. There are several monounsaturated fatty acids that occur naturally with double bonds at positions other than the ninth carbon of the carboxyl group of fatty acid. For example, the triacylglycerols of Limnanthes alba and many other gymnosperms contain monounsaturated fatty acids with a double bond at the delta-5 position. This activity can be catalyzed by a delta-5 desaturase which, unlike delta-9 desaturase, which uses 18: l-CoA as a substrate for the desaturation reaction, can instead use 20: 0-CoA (Pollard, MR and Stumpf, PK (1980) Plant Physiol 66: 649-655; Moreau, RA et al. (1981) Arch Biochem Biophys 209: 376-384). Prairie foam (Limnanthes alba) is a plant native to higher elevations of Northern California and Southern Oregon. The triacylglycerol fraction of mature seed is composed mainly of fatty acids containing 20 or 22 carbons and one or two double bonds (20: 1, 22: 1 and 22:20). This double bond is unusual in so far as It is in a position not normally found in the fatty acids of common plant oils: in the delta-5 position.The Limnanthes elongase seems to prefer palmitoyl-CoA (16: 0-CoA) as its substrate instead of oleoyl-CoA ( 18: 1 delta-9-CoA), the common substrate for known vegetable fatty acid elongases In Limnanthes, 16: 0 CoA is extended to 20: 0-CoA and is desaturated at 20: 1 delta-5. the formation of 20: 1 delta-11 as the arabidopsis or nabine, where 18: 1 delta-9 is extended to 20: -l delta-11 (Pollard, M. R. and Stumpf, P. K. (1980) Plant Physiol 66: 649-655). The genes encoding Limnanthes delta-5 desaturase and the fatty acyl elongase functions to date have not been isolated and are subject of the present application. Although most plants contain at least minimal amounts of long chain fatty acids, FAS is not involved in the de novo production of these very long chain fatty acids. Instead, the FAS product is exported from the plastid and converted to acyl-CoA derivatives, which then serve as substrates for the fatty acid elongation system (FAE). The gene involved in FAE of Arabidopsis has been located in the FAE1 locus. Jojoba oil consists mainly of waxes which are esters of monounsaturated fatty acids and alcohols, most of which contain fatty acid chains with more than 18 carbons. The elongation to form very long chain fatty acids in Arabidopsis, jojoba and rapeseed uses malonyl-CoA and acyl-CoA as substrates (Lassner, M. et al. (1996) Plant Cell 8: 281-292). In Limnanthes biosynthesis of fatty acids 20: 0 is predominantly produced by elongation of the palmitate chain as the initial substrate; therefore, the enzyme that catalyzes this reaction must be similar, although different from the enzyme involved in the production of very large chain fatty acids through the elongation of malonyl-CoA.

The ability to manipulate fatty acid biosynthesis pathways by genetic engineering will also allow changes to the fatty acid composition of vegetable oils and / or introduce entirely new pathways into oilseeds to produce novel biopolymers from acetyl- CoA. The oils of Limnanthes and fatty acids have potential use as industrial agents. The stolides are oligomeric fatty acids containing a secondary ester bond in the alkyl main structure of the fatty acids. The delta-5 20: 1 fatty acids present in Limnanthes oil are useful for the production of polystolides wherein the single delta-5 bond stabilizes the compound (Isbell, TA and Kleiman, R. (1996) J Am Oil Chem Soc 73: 1097-1107). The biodegradation of polyunsolides derived from monounsaturated fatty acids from Limnanthes appears to be slower than the biodegradation of polystyides derived from soybean oils or oleic oils, but the biodegradation continues over time so that all the stolides are likely to eventually degrade in the nature (Ehran, SM and Kleiman, R. (1997) J Am Oil Chem Soc 74: 605-606). This resistance to bacterial degradation suggests that polystyides derived from 20: 1 delta-5 fatty acids will produce lubricants, fats, plastics, inks, cosmetics and surfactants with a long shelf life.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to isolated fragments of nucleic acid encoding the Limnanthes oil biosynthetic enzymes. Specifically, this invention relates to an isolated fragment of nucleic acid encoding a delta-5 acyl-CoA desaturase or an acyl-CoA elongase fat. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding a delta-5-acyl-CoA desaturase or an acyl-CoA elongase fat. The extension of 16: 0-CoA to 20: 0 is also described by the acyl-CoA elongase fat from Limnanthes. We also show that delta-5 desaturase, in the absence of 20: 0 -CoA, will insert a double bond at position delta-5 of 16: 0 and 18: 0-CoA. A further embodiment of the present invention pertains to a polypeptide that codes for all or a substantial portion of an enzyme involved in lipid biosynthesis, which is selected from the group consisting of a delta-5-acyl-CoA desaturase and an acyl -CoA elongasa fat. In another embodiment, the present invention relates to a chimeric gene encoding a delta-5 acyl -CoA desaturase or an acyl -CoA elongase fat, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a fragment of nucleic acid coding for delta-5 acyl-CoA desaturase or an acyl-CoA elongase fat, operably linked to suitable regulatory sequences, wherein the expression of the chimeric gene results in the production of the encoded protein in a transformed host cell. In a further embodiment, the present invention relates to a transformed host cell comprising in its genome a chimeric gene coding for delta-5 acyl-CoA desaturase or a fatty acyl-CoA elongase, operably linked to suitable regulatory sequences. The expression of the chimeric gene with respect to the production of the protein encoded in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and includes cells derived from higher plants and microorganisms. The invention also includes transformed embryos and plants arising from transformed host cells of higher plants, and seeds derived from such transformed plants. A further embodiment of the present invention relates to a method for altering the level of delta-5 acyl-CoA desaturase or an acyl -CoA elongase fat in a transformed host cell, comprising: a) transforming a host cell with a chimeric gene which comprises a nucleic acid fragment encoding delta-5 acyl -CoA desaturase or an acyl-CoAl elongase fat and b) growing the transformed host cell under conditions that are suitable for the expression of the chimeric gene, wherein the expression of the gene chimeric results in the production of altered levels of delta-5 acyl-CoA desaturase or an acyl-CoA elongase fat in the transformed host cell. A further embodiment of the present invention relates to a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a delta-5 acyl-CoA desaturase or an acyl-CoA elongase grease. In a further embodiment, the present invention relates to a method for producing a desaturated fatty acid, comprising a double bond at the delta-5 position in a host cell, and seeds, oils and methods for producing seed oils wherein the Seeds and oils comprise a desaturated fatty acid wherein the fatty acid comprises a double bond at the delta-5 position. A further embodiment of the present invention is a method for reducing the level of fatty acids of 16 carbons in a host cell, and a method for increasing the level of fatty acids of 20 carbons in a host cell, and seeds, oils and methods to produce seed oils with reduced levels of 16-carbon fatty acids or increased levels of 20-carbon fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS AND DESCRIPTION OF THE SEQUENCES The invention can be more fully understood from the following detailed description and the accompanying drawings and in the sequence listing, which form a part of this application. Figure 1 shows the pathways for the formation of long chain fatty acids found in Limnanthes seeds. The biosynthesis of palmitate (16: 0), stearate (18: 0) and oleate (18: 1) is produced in the plastid, while the lengthening of palmitate to araquinodate and delta-5 desaturation occurs in the endoplasmic reticulum ( adapted from Pollard, MR and Stumpf, PK (1980) Plant Physiol 66: 649-655). Figure 2 shows an alignment of the amino acid sequences of the delta-9 desaturase from Arabidopsis thaliana (SEQ ID NO: 3) and the delta-5 acyl-CoA desaturase from current Limnanthes (Ide .pk008.b9; DE IDENT NO: 2). The amino acids which are identical in both sequences are marked with an asterisk (*) above the alignment. The scripts are used by the program to maximize the alignment of the sequences. Figure 3 shows the traces of gas chromatograms obtained for the oils of wild-type soybean embryos (Figure 3 (A)) and the soybean embryos expressing the acyl-CoA elongase fat from Limnanthes (Figure 3 (B)), which demonstrate the production of C20 fatty acids in processed soybean embryos. The fatty acids corresponding to the various peaks of the chromatogram are indicated. Figure 4 shows the decrease in 16: 0 fatty acid accumulation concomitant with the increase in 20: 0 fatty acids in transgenic bean embryos that express acyl-CoA elongase Limnanthes fat. Figure 5 shows the traces from gas chromatograms obtained for the oils of wild-type soy bean embryos (Figure 5 (A)) and soybean embryos expressing the delta-5 acyl-CoA desaturase Limnanthes (figure 5 (B)). The relevant fatty acids are indicated by their retention time: 2.209 is 16: 0; 2.271 is 16.-1? 5; 3,477 is 18: 0; 3.530 is 18: l? 5; and 3,567 is 18:? 9. Figure 6 shows GC-MS analysis of fatty acid methyl esters prepared from soy bean embryos expressing delta-5 acyl-CoA desaturase from Limnanthes demonstrating the formation of fatty acids 16: 1 delta-5 and 18: 1 delta-5. Figure 6 (A) represents the gas chromatogram in which MDDS derivatives of methylhexanediocene acid are identified using ion scouting selected for 362 m / z. Figure 6 (B) is the mass spectrum of the largest of the two peaks evident in Figure 6 (A). Figure 6 (C) depicts the gas chromatogram wherein the DMDS derivatives of methyloctadecenoic acid are identified using an ion scan selected at 390 m / z. Figure 6 (D) is the mass spectrum of the front flange of the largest peak that is evident in Figure 6 (C). The following descriptions of the sequences and sequence listings appended hereto comply with the rules governing descriptions of nucleotide or amino acid sequences in patent applications, as set forth in 37 C.F.R. §1.821-1.825. The SEC. FROM IDENT. NO: 1 is a nucleotide sequence comprising the entire cDNA insert in the Ide .pkOOOd .b9 clone encoding the entirety of the delta-5 acyl-CoA desaturase from Limnanthes. The SEC. FROM IDENT. NO: 2 is a deduced amino acid sequence of a delta-5-acyl-CoA desaturases from Limnanthes derived from the nucleotide sequence of SEC. FROM IDEJSTT. NO: 1. The SEC. FROM IDENT. NO: 3 is the amino acid sequence of the delta-9 desaturase of Arabidopsis thaliana having a general identifier NCIB number: 2970034.

The SEC. FROM IDENT. NO: 4 is the nucleotide sequence that comprises the contiguous assembly from the cDNA insert in the Ide .pkOOOd clones. d5 and Ide.pk0015.dl0 that encode Xa totality of the acyl-CoA elongase fat from Limnanthes. The SEC. FROM IDENT. NO: 5 is the deduced amino acid sequence of an acyl-CoA elongase complete limnanthes fat derived from the nucleotide sequence of the SEC. FROM IDENT. NO: 4 The SEC. FROM IDENT. NO: 6 is the nucleotide sequence comprising a portion of the cDNA insert in the Ide .pkOOlO clone. e4 that codes for a portion of the acyl-CoA elongase fat from Limnanthes. The SEC. FROM IDENT. NO: 7 is the deduced amino acid sequence of a portion of the acyl-CoA elongase fat from Limnanthes derived from the nucleotide sequence of the SEC. FROM IDENT. NO: 6. The sequence listing contains the one-letter code for nucleotide sequence characters and the three-letter codes for amino acids as identified in accordance with the IUPAC-IUBMB standards described in Nucleic Acids Research 13: 3021-3030 (1985) and in the Biochemical Journal 219 (No. 2): 345-373 (1984) which are incorporated herein by reference. The symbols and formats used for the nucleotide and amino acid sequence data comply with the rules established in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION In this context of this description, numerous terms will be used. As used herein, an "isolated fragment of nucleic acid" is an RNA or DNA polymer having single or double chain, and optionally containing synthetic, non-natural or altered nucleotide bases. An isolated fragment of nucleic acid in the form of a DNA polymer can be constituted of one or more segments of cDNA, genomic DNA or synthetic DNA. As used herein, the term "contiguous" refers to an assembly of overlapping nucleic acid sequences to form a contiguous nucleotide sequence. For example, several DNA sequences can be compared and aligned to identify common or overlapping regions. The individual sequences can then be assembled into a single contiguous nucleotide sequence. As used herein, the term "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases result in the substitution of one or more amino acids, but which do not affect the functional properties of the nucleic acid. protein encoded by the DNA sequence. The term "substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate the alteration of gene expression by antisense or co-suppression technology. The term "substantially similar" also refers to modifications of the nucleic acid fragments of the present invention such as the deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript versus the ability to mediate the alteration. of the expression of the gene by antisense or cosuppression technology or alteration of the functional properties of the resulting protein molecule. Therefore, it is understood that the invention encompasses more than the exemplary specific sequences. For example, it is well known in the art that antisense suppression and co-suppression of gene expression can be accomplished by using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments. that do not share 100% identity of the sequence with the gene to be deleted. In addition, alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but which do not alter the functional properties of the encoded protein, are well known in the art. Therefore, a codon for the amino acid alanine, a hydrophobic amino acid, can be replaced by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue such as valine, leucine or isoleucine. Similarly, changes that result in the substitution of a negatively charged residue for another, such as aspartic acid for glutamic acid or another positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. . The nucleotide changes which result in the alteration in the N-terminal or C-terminal portions of the protein molecule are also not expected to alter the activity of the protein. Each of the proposed modifications is within the usual skill in the art, since it is the determination of biological activity retention of the coded products. In addition, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize, under astringent conditions (0.1X SSC, 0.1% SDS, 65 ° C), with nucleic acid fragments described herein. The substantially similar nucleic acid fragments of the present invention can also be characterized by the percent similarity of the sequence of amino acids they encode, with the amino acid sequences described herein, determined by algorithms commonly used by those skilled in the art. technique. Preferred are those nucleic acid fragments whose nucleotide sequences encode for amino acid sequences that are 80% similar to the amino acid sequences reported herein. The most preferred nucleic acid fragments encode for amino acid sequences that are 90% similar to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are 95% similar to the amino acid sequences reported herein. Sequence alignments and percent similarity calculations are performed using the Megalign program of the LASARGENE bioinformatics computer (DNASTAR Inc., Madison, Wl). The multiple alignment of the sequences can be done using the Clustal method of alignment (Higging, DG and Sharp, PM (1989) CABIOS 5: 151-153) with the parameters that are set by default (SEPARATION PENALTY = 10, PUNISHMENT OF SEPARATION LENGTH = 10). The default parameters for paired alignments using the Clustal method are KTUPLE 1, SEPARATION PUNISHMENT = 3, INTERVAL = 5 and SAVED DIAGONAL = 5. A "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or nucleotide sequence of a gene to provide putative identification of that polypeptide or gene, either by manual evaluation of the sequence by a person skilled in the art, or by an automated sequence comparison on computer and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S.F., et al., (1993) J. Mol. Biol. 215: 403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or 30 or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. In addition, with respect to the nucleotide sequences, gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides can be used in sequence dependent or gene identification methods (e.g., Southern hybridization) and isolation (e.g., in situ colony hybridization). bacterial or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases can be used as PCR amplification primers in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises sufficient of the sequence to provide specific identification and / or isolation of a nucleic acid fragment comprising the sequence. The present specification describes partial or complete amino acid or nucleotide sequences that code for one or more particular plant proteins.

Those skilled in the art who have the benefit of the sequences as presented herein may now utilize all or a substantial portion of the described sequences for purposes known to those skilled in the art. Accordingly, the present invention comprises the complete sequences as presented in the attached sequence listing, as well as substantial portions of those sequences as defined above. The term "codon degeneracy" refers to a divergence in the genetic code that allows variation in the nucleotide sequence without altering the amino acid sequence of a coded polypeptide. Accordingly, the present invention relates to any nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding delta-5 acyl-CoA desaturase of the fatty acyl-CoA elongase proteins, as establishes in the SEC. FROM IDENT. N0: 2, 5 and 7. Those skilled in the art are well aware of the "codon displacement" shown by specific host cells in use of nucleotide codons to specify a given amino acid. Therefore, when a gene for enhanced expression is synthesized in a host cell, it is desirable to design the gene so that its codon usage frequency approximates the frequency of use of preferred codons of the host cell.

"Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are linked and reassociated to form gene segments which are then assembled enzymatically to build the complete gene. The term "chemically synthesized" insofar as it is related to a DNA sequence means that the component nucleotides are assembled in vitro. Manual chemical synthesis of DNA can be carried out using well-established procedures, or automated chemical synthesis can be carried out using one of numerous commercially available machines. Accordingly, genes can be adapted for optimal gene expression based on nucleotide sequence optimization to reflect the deviation of the host cell codon. Those skilled in the art appreciate that the probability of success of gene expression if codon deviation towards these codons is used, is favored by the host. The determination of the preferred codons can be based on an investigation of the genes derived from the host cell in which the sequence information is available. The term "gene" refers to a nucleic acid fragment that expresses a specific protein, which includes regulatory genes that precede (5 'non-coding sequences) and that follow (3' non-coding sequences) the coding sequence. The term "native gene" refers to a gene as found in nature with its own regulatory sequences. The term "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature. The term "endogenous gene" refers to a native gene in its natural position in the genome of an organism. A "foreign gene" refers to a gene that is not normally found in the host organism, but is introduced into the host organism by gene transfer. Foreign genes can include native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the gemoma by a transformation procedure. The term "coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. The term "regulatory sequences" refers to nucleotide sequences located towards the 5 'end (5' non-coding sequences) within, or towards the 3 'end (3' non-coding sequences) of a coding sequence, and which alter the transcription, processing or stability of the RNA, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns and polyadenylation recognition sequences. The term "promoter" refers to DNA sequences capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3 'to a promoter sequence. The promoter sequence consists of proximal and matistal elements towards the 5 'end, these latter elements are often referred to as extensions. Accordingly, an "extender" is a DNA sequence which can stimulate the activity of the promoter and can be an innate element of the promoter or can be a heterologous element inserted to improve the level of tissue specificity of a promoter. The promoters can be derived in their entirety from a native gene, or can be made up of different elements derived from different promoters found in nature, or even comprising synthetic segments of DNA. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters, which cause a gene to be expressed in most cell types most of the time, are commonly referred to as "constitutive promoters". New promoters of various types, useful in plant cells, are constantly being discovered; many examples can be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15: 1-82. It is further recognized that since in most cases the exact boundaries of the regulatory sequence have not been defined, DNA fragments of different lengths may have identical promoter activity. The term "translation leader sequence" refers to a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA towards the 5 'end of the translation start sequence. The translation leader sequence may affect the processing of the primary transcript to mRNA, the stability or the translation efficiency of mRNA. Examples of translation leader sequences have been described (Turner, R. and Foster, G.D. (1995) Molecular Biotechnology 3: 225). The term "3 'non-coding sequences" refers to DNA sequences located towards the 3' end of a coding sequence and includes polyadenylation recognition sequences and other sequences that encode regulatory signals capable of affecting mRNA processing or expression of genes. The polyadenylation signal is usually characterized in that it affects the addition of the polyadenylic acid tract to the 3 'end of the mRNA precursor. The use of different non-coding sequences 31 is exemplified by Ingelbrecht et al., (1989) Plant Cell 1: 671-680. The term "RNA transcript" refers to the product that results from transcription catalyzed by RNA polymerase 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 can be an RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as mature RNA. The term messenger RNA "mRNA" refers to RNA that is found without introns and that can be translated into the protein by the cell. The term "cDNA" refers to a double-stranded DNA that is complementary and that is derived from mRNA. The term "forward" (meaningful) RNA refers to an RNA transcript that includes the mRNA and that must be translated into the protein by the cell. The term "antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of an objective gene (U.S. Patent No. 5,107,065, incorporated herein by reference) . The complementarity of an antisense RNA can be in any part of the transcribed gene ie in the 5 'non-coding sequence, the 3' non-coding sequence, introns or in the coding sequence. The term "functional RNA" refers to direct RNA, antisense RNA, ribosome RNA or other RNA that may not be translated but still has an effect on cellular processes. The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of altering the expression of a coding sequence - (i.e., that the coding sequence is under the transcriptional control of the promoter). The coding sequences can be operably linked to regulatory sequences in direct or antisense orientation. The term "expression" as used herein refers to the transmission and stable accumulation of direct (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. The expression can also refer to the translation of mRNA into a polypeptide. The term "antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. The term "overexpression" refers to the production of a gene product in transgenic organisms that exceed production levels in normal or non-transformed organisms. The term "cosuppression" refers to the production of direct RNA transcripts capable of suppressing the expression of substantially similar foreign or endogenous genes (U.S. Patent No. 5)., 231,020, incorporated herein by reference). The term "altered levels" refers to the production of the product. or gene products in transgenic organisms in amounts or proportions that differ from those of normal or untransformed organisms. A "mature" protein refers to a polypeptide processed post-translationally; that is, one from which the pre- or propeptides present in the primary translation product have been removed. The "precursor" protein refers to the primary product of mRNA translation; that is, with pre- and propeptides still present. The pre- and pro-peptides may be, but are not limited to intracellular localization signals. _ A "chloroplast transit peptide" is an amino acid sequence which is translated together with a proton and directs the protein to the chloroplasts or other types of plastids present in the cell in which the protein is produced. The term "chloroplast transit sequence" refers to a nucleotide sequence that codes for a chloroplast transit peptide. A "signal peptide" is an amino acid sequence which is translated together with a protein and directs the protein to the story system (Chrispeels, JJ, (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42: 21-53 ). If the protein is to be directed to a vacuole, a vacuolar direction signal (supra) can be added additionally, or if it is towards the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) can be added. If the protein is to be directed to the nucleus, any signal peptide present must be removed instead of this a nuclear localization signal must be included (Raikhel (1992) Plant Phys. 100: 1627 -163-2). The term "transformation" refers to the transfer of a nucleic acid fragment within the genome of a host organism, resulting in genetically stable inheritance. Host organisms that contain transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of plant transformation methods include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol 143: 277) and accelerated particle or "gene gun" transformation technology (Klein TM et al. (1987) Na ture (London) 327: 70-73; US Patent No. 4, 945, 050, incorporated herein by reference). The standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are more fully described in Sambrook, J., Fritsch EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis"). Nucleic acid fragments that code for at least a portion of two enzymes involved in lipid biosynthesis have been isolated and identified by comparison of random plant sequences of cDNA with public database having nucleotide and protein sequences using BLAST algorithms well known to those skilled in the art The identity of these enzymes has been confirmed by functional analysis, as set out in example 6. Table 1 includes the proteins described herein, and the designation of the cDNA clones comprising the acid fragments or nucleic acid that code for these proteins.

TABLE 1 Biosynthetic Enzymes of Limnanthes Oil Enzyme clone Plant Delta-5-acyl-CoA desaturase Ide.pk0008.b9 Limnanthes douglasii Acyl-CoA contiguous fat elongase from Limnanthes douglasii Ide.pk0008d5 Ide.pk0015.d l0 Ide.pk0010.e4 Limnanthes douglasii The nucleic acid fragments of the present invention can be used to isolate cDNAs and genes that code for homologous proteins from the same species or from other plant species. Isolation of homologous genes used in sequence dependent protocols are known in the art. Examples of sequence dependent protocols include, but are not limited to, nucleic acid hybridization methods and DNA and RNA amplification methods, as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction , ligase chain reaction). For example, genes encoding other delta 5-acyl-CoA desaturase or acyl-CoA elongase fat homologs, either cDNA or genomic DNA, can be isolated directly by utilizing all or a portion of the present fragments of nucleic acid as DNA hybridization probes to analyze libraries of any desired plant using methodology well known to those skilled in the art. Oligonucleotide-specific probes based on the present nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). further, all of the sequences for synthesizing DNA probes can be used by methods known to those skilled in the art such as random labeling of DNA primer, nick translation, or end labeling techniques, or probe probes. RNA using available in vitro transcription systems. In addition, specific primers can be designed and can be used to amplify some or all of the present sequences. The resulting amplification products can be directly labeled during the amplification fractions or can be labeled after the amplification reactions, and can be used as probes to isolate full-length cDNAs or genomic fragments under appropriate stringency conditions. In addition, two short segments of the present nucleic acid fragments can be used in polymerase chain reaction protocols to amplify larger fragments of nucleic acid encoding homologous genes from DNA or RNA. The polymerase chain reaction can also be performed in a library of cloned fragments of nucleic acid wherein the sequence of a primer is derived from the present nucleic acid fragments, and the sequence of another primer takes advantage of the presence of the acid tracts polyadenylic towards the 3 'end of the precursor mRNA encoding plant genes. Alternatively, the second primer sequence can be based on the sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et al., (1988) Proc? Nati. Acad. Sci. USA 85: 8998) to generate cDNA by using PCR to amplify copies of the region between a single point in the transcript and at the 3 'or 5' end. The primers oriented in the 3 'and 5' directions can be designed from the present sequences. Using commercially available 3 'RACE or 5' RACE systems (BRL), specific 3 'or 5' cDNA fragments can be isolated (Ohara et al., (1989) Proc. Na ti. Acad. Sci. USA 86: 5673; Loh et al., (1989) Science 243: 217). The products generated by the 3 'and 5' RACE procedures can be combined to generate CDNA of full lengths (Frohman, M.A. and Martin, G.R., (1989) Techniques 1: 165). The availability of the present nucleotides and the deduced amino acid sequences facilitate the immunological analysis of cDNA expression libraries. Synthetic peptides representing portions of the present amino acid sequences can be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins, comprising the amino acid sequences. These antibodies can then be used to analyze cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner, R.A. (1984) Adv. Immuno 1. 36: 1; Maniatis). The biosynthesis of oil in plants has been studied in depth (see Harwood (1989) in Critical Reviews in Plant Sciences, Vol. 8: 1-43). As used herein, "oilseed crops" refers to plant species which produce and store triacylglycerol in specific organs, primarily seeds. In particular, for purposes of this description, the term "oilseed crops" refers to soybean, corn, sunflower, peanut, safflower, sesame, niger, cotton, cocoa, flax seed (flax), low linoleic flax. , curing oil, palm oil, coconut oil, nabine oil and other oilseed species of Brassica such as B. napus, B. campestris, B. Smell cea, B. carinata, B. Júncea, B. nigra, B. adpressa, B. tournefortii, B. fruticulosas. The nucleic acid fragments of the present invention can be used to create transgenic plants in which delta 5-acyl-CoA desaturase or acyl-CoA elongase fat is present at levels higher or lower than normal or in cell types or stages of development in which they are not normally found. This would have the effect of altering the saturation level of fatty acid and the chain length in these cells. As demonstrated in experiment 6 below, overexpression of the delta 5-acyl-CoA elongase Limnanthes fat in an oilseed crop results in the elongation of palmitic acid (16: 0) to arachidonic acid (20: 0). The overexpression of Limnanthes delta 5-acyl-CoA desaturase in an oilseed crop results in the introduction of a double bond at the 5-acyl delta position of a fatty acid chain, resulting in the production of delta fatty acids -5 16: 1 and 18: 1. Overexpression of both genes in an oilseed crop will allow the production of delta-5 20: 1 fatty acids. There are at least two positive effects that arise from this: the reduction of saturated fatty acids (especially 16: 0) in edible oils and the production of fatty acids (20: 1 delta-5) with a large number of industrial uses. The overexpression of the delta 5-acyl-CoA desaturase or fatty acyl-CoA elongase proteins of the present invention can be carried out by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing the expression of a gene in the desired tissues and in the stage of development that is desired. For reasons of convenience, the chimeric gene may be comprised of promoter sequences and translation leader sequences derived from the same genes. It is also possible to provide 3 'non-coding sequences coding for transcription termination signals. The present chimeric gene may also comprise one or more introns in order to facilitate the expression of the gene. The plasmid vectors comprising the current chimeric gene can then be constructed. The choice of plasmid vector will depend on the method that will be used to transform host plants. Those skilled in the art will understand that genetic elements must be present in the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. A person skilled in the art will also recognize that different independent transformation phenomena will result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4: 2411-2418; De Almeida et al., (1989) Mol. Gen Genetics 218: 78-86), and therefore that multiple phenomena must be analyzed in order to obtain lines that show the level and expression pattern desired. Such analysis can be carried out by Southern analysis or DNA, Northern or mRNA expression analysis, Western analysis or protein expression, or phenotypic analysis. For some applications it may be useful to direct the present enzyme involved in lipid biosynthesis to different cell compartments, or to facilitate its secretion from the cell. It is therefore considered that the chimeric gene described above can be further supplemented by altering the coding sequence to encode a delta 5-acyl-CoA desaturase or an acyl-CoA elongase fat with appropriate extracellular target sequences such as transit sequences ( Keegstra, K. (1989) Cell 56: 247-253), signal sequences or sequences coding for localization in the endoplasmic reticulum (Chrispeels, JJ, (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42 : 21-53), or nuclear localization signals (Raikhel, N. (1992) Plant Phys 100: 1627-1632) aggregated and with target sequences that have been removed beforehand. Although the aforementioned references provide examples of each of these, the list is not exhaustive and more directional signals may be discovered in the future that may be useful. The present delta 5-acyl-CoA desaturase or acyl-CoA elongase fat (or portions thereof, in heterologous host cells, particularly in the cells of microbial hosts, can be produced and can be used to prepare antibodies for these proteins by methods Well known to those skilled in the art Antibodies are useful for detecting delta 5-acyl-CoA desaturase or acyl-CoA fat elongase in situ in cells, or in vitro in cell extract The preferred heterologous host cells for production of the delta 5-acyl-CoA desaturase or current acyl-CoA elongase fat are microbial hosts Microbial expression systems and expression vectors that contain regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these can be used to build a chimeric gene for the production of the current delta 5 Acyl-CoA desaturase or acyl-CoA elongase fat. This chimeric gene must then be introduced into appropriate microorganisms via transformation to provide a high level of expression of the encoded enzyme involved in lipid biosynthesis. An example of a vector for a high expression level of the current 5-acyl-CoA desaturase or acyl-CoA elongase fat in a bacterial host is provided (example 7). All or a substantial portion of the nucleic acid fragments of the present invention can also be used as probes to genetically and physically map the genes to which they are a part, and as markers for traits attached to those genes. Such information can be useful in plant breeding in order to develop lines with desired phenotypes. For example, current fragments of nucleic acid can be used as restriction fragment length polymorphism (RFLP) markers, can be probed by Southern blotting (Maniatis) of restriction-digested plant genomic DNA with nucleic acid fragments of the present invention. The resulting band patterns can then be subjected to genetic analysis using computer programs such as MapMaker (Lander et al, (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the present invention can be used to perform Southern blot tests containing genomic DNAs treated with restriction endonucleases from a set of individuals representing the father and progeny of a defined genetic cross. The segregation of DNA polymorphisms is noted, and is used to calculate the possession of the current nucleic acid sequence in the genetic map previously obtained using this population (Botstein, D., et al., (1980) Am, J. Hum. Genet. 32: 314-331 ). The production and use of probes derived from plant genes for use in genetic mapping is described in R. Bernatzky, R and Tanksley, S. D., (1986) Plant Mol. Biol. Repórter 4 (1).-37-41. Many publications describe the genetic mapping of specific cDNA clones using the methodology indicated above or variations thereof. For example, the crossing of F2 populations, backcross populations, randomly matched populations, almost isogenic lines and other sets of individuals can be used for mapping. Such methodologies are well known to those skilled in the art. Nucleic acid probes derived from current nucleic acid sequences can also be used for physical mapping (ie, placement of sequences on physical maps).; see Hoheisel, J. D., et al., In: Nonmammalian Genomic Analysis: A Practical Guide, Academic Press 1986. pp. 319-346 and references that are mentioned there. In another embodiment, nucleic acid probes derived from current nucleic acid sequences can be used in the direct fluorescence in situ hybridization (FISH) mapping (Trask, B. J. (1991) Trends Genet 7: 149-154). Although current methods of FISH mapping favor the use of large clones (from several to several hundred KB, see Laan, M. et al., (1995) Genome Research 5: 13-20), an improvement in sensitivity can allow the operation of the mapping by FISH using shorter probes. Various methods based on nucleic acid amplification of a genetic and physical mapping can be carried out using the current nucleic acid sequences. Examples include allele-specific amplification (Kazazian, HH (1989). "LaD.Clin.Med 114: (2) - .95-96), polymorphism of genes amplified by PCR (CAPS; Sheffield, VC et al., 1993 ) Genomics 16: 325-332), allele-specific ligation (Landegren, U. et al. (1988) Science 241: 1077-1080), nucleotide extension reactions (Sokolov, BP (1990) Nucleic Acid Res. 18: 3671), radiation hybridization mapping (Walter, MA et al (1997) Nature Genetics 7: 22-28) and Happy mapping (Dear, PH and Cook, PR (1989) Nucleic Acids Res. 17: 6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce pairs of primers for use in the amplification reaction or primer extension reactions The design of such primers is well known to those skilled in the art. In the methods that use genetic mapping based on PCR, it may be necessary to identify different DNA counts between the parents of a mapping cross in the region corresponding to the current nucleic acid sequence. However, this is generally not necessary for mapping methods.

EXAMPLES The present invention is further defined by the following examples, in which all parts and percentages are by weight, and degrees by Celsius, unless otherwise indicated. It should be understood that these examples, while indicating preferred embodiments of the invention, are provided by way of illustration only. From the previous discussion of these examples, a person skilled in the art can determine the essential characteristics of the invention, and without departing from the spirit and scope of the same can make various changes and modifications of the invention to adapt it to the various uses and conditions.

EXAMPLE 1 Composition of cDNA library: Isolation and sequencing of cDNA clones CDNA libraries representing the mRNAs of embryonic tissues of Limnanthes douglasii are prepared in pcDNAlI vectors, according to the manufacturer's protocol (Invitrogen Corporation, Carlasbd, CA). CDNA inserts are amplified from randomly taken bacterial colonies containing the pcDNAII recombinant plasmids via polymerase chain reaction using primers specific for the vector sequences flanking the inserted cDNA sequences or plasmid DNA and prepared from cells bacterial cultures. The amplified insert DNAs or the plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (which express sequence tags or "EST", see Adams, MD et al., (1991) Science 252: 1651). The resulting ESTs are analyzed using a Perkin Elmer model 377 fluorescent sequencer.

EXAMPLE 2 Identification of cDNA clones The enzymes that code for the ESTs involved in lipid biosynthesis are identified by carrying out the BLAST test (Basic Local Alignmet Search Tool; Altschul, SF, et al., (1993) J. Mol. Biol. 215: 403- 410, see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to the sequences contained in the "nr" BLAST database (which includes all non-redundant CDB GenBank translations, sequences derived from of the Brookhaven Protein Data Bank three-dimensional structure, the latest major publications of the SWISS-PROT protein sequence database, the EMBL and DDBJ databases). The cDNA sequences obtained in Example 1 are used by similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences are translated in all reading frames and compared for similarity with all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish, W. and States, DJ (1993) Nature Genetics 3: 266-272) provided by NCBI. For convenience, the P value (probability) of observing a match of a cDNA sequence with a sequence contained in the databases investigated solely by chance as calculated by BLAST is reported herein as values of "pLog" which represents the negative of the logarithm of the P value reported. Consequently, the higher the pLog value, the greater is the probability that the cDNA sequence and BLAST "agree" on the representation of homologous proteins.

EXAMPLE 3 Characterization of cDNA clones that code for delta-5-acyl-CoA desaturase homologs The BLASTX research using the EST sequences from the clones Ide.pk0004.cl0, Ode .pk0012. e5 e Ide.pk0012.gil and the entire cDNA insert from the Ide .pk0010 clone. a8, show similarity of the proteins encoded by the cDNA with delta-9-acyl-desaturase lipid / delta-9-acyl-CoA desaturase from Rosa hybrida (GenBank accession number S80863, general identifier NCBI number 1911477). The investigation with BLASTX using the EST sequence from the clone pk.0008.b9 shows similarity of the protein encoded by the cDNA with the fatty acid desaturase of Rosa hybrida (GenBank access number D49383; NCBI general identifier 2580425). Table 2 shows the BLAST results of each of these sequences.

TABLE 2 BLAST results for clones encoding homologous to desaturated polypeptides clone GenBank access number rating score pLog BLAST Ide.pk0004.clO S80863 48.23 Ide.pk00012.e5 S80863 23.64 Ide.pk0012.gll S80863 14.42 Ide.pk0010.a8 S80863 9.89 Ide.pk0008.b9 D49383 1.44"_ The sequence of the entire cDNA insert Ide .pkOOOd .b9 is determined and shown in SEQ. FROM IDENT. NO: l; the deduced amino acid sequence of this cDNA is shown in SEQ. FROM IDENT. NO: 2 The EST sequences for the clones Ide.pk0004.cl0, Ide .pk0012.e5, Ide.pk0012.gil and Ide.pk0010.a8 are included by the sequence established in SEC. FROM IDENT. NO: l. The amino acid sequence established in the SEC is evaluated. FROM IDENT. NO: 2 by BLASTP ", which provides a pLog value of> 250 versus the delta-9 desaturase sequence of Arabidopsis thaliana (general identifier NCBI number 2970034) Figure 1 represents an alignment of the amino acid sequences that are established in SEQ ID NO: 2 and the delta-9 desaturase sequence of Arabidopsis thaliana (SEQ ID NO: 3) The amino acid sequence set forth in SEQ ID NO: 2 is 47.9% similar to the sequence of Arabidopsis thaliana (SEQ ID NO: 3) Sequence alignments and percent identity calculations are performed using the Megalign program of LASARGENE bioinformatic calculation equipment (DNASTAR Inc., Madison , Wl) Paired alignment of the amino acid sequences and percent similarity calculations are performed using the Clustal alignment method (Higgins, DG and Sharp, PM (1989) CABIOS 5: 151-153) with parameters for omission (PENALTY OF SEPARATION N = 5, K TUPLO = 1, INTERVAL = 5 and DIAGONALS SAVED = 5). BLAST scores, sequence alignments and probabilities suggest that the current nucleic acid fragment codes for the entire delta-9-acyl-CoA desaturase from Limnanthes. However, oil derived from Limnanthes is mainly composed of very long chain fatty acids with a delta-5 double bond, which suggests that the current nucleic acid fragment can indeed code for delta-5-acyl-CoA desaturase in instead of doing it for delta-9 desaturase. As shown in example 6, the expression of Limnanthes desaturase in soy bean embryos results in the formation of oils containing delta-5 fatty acids 16: 1 and 18: 1. Accordingly, the current nucleic acid fragments comprise the first sequences of Limnanthes douglasii encoding delta-5 acyl-CoA desaturase.

EXAMPLE 4 Characterization of cDNA clones that code for acyl-CoA elonase fatty acid homologs The BLASTX search using the EST sequences from the clones Ide.pk0008d5 and Ide .pkOOlO. e4 show similarity of the proteins encoded by the cDNAs for beta-ketoacyl-CoA synthase from Arabidopsis thaliana GenBank accession number AC00310-5: NCBI general identifier number 2760830). In Table 3, the BLAST results of these ESTs are shown: TABLE 3 BLAST results for a clone encoding a polypeptide homologous to beta-ketoacyl-CoA synthase PLog score BLAST Clone AC003105 Ide.pk0008.d5 78.05 Ide.pkOOlO. e4 72.66 Further investigation of the patented database indicates that the clone Ide.pk0015.dl0 also shows similarities with beta-ketoacyl-CoA synthase. The sequence of the entire cDNA insert in the clone Ode.pkOOOd was determined. d5 and a contiguous with this sequence and the sequence was assembled from a portion of the cDNA insert from the clone Ide.pk0015.dl0. The nucleotide sequence of this contiguous is shown in SEC. FROM IDENTX NO: 4; the deduced amino acid sequence of this contiguous is shown in SEC. FROM IDENT. NO: 5. A BLASTX search using the nucleotide sequences established in the SEC. FROM IDENT. NO: 4 results in a pLog value of <; 254 versus the beta-ketoacyl-CoA synthase sequence of Arabidopsis thaliana. The sequence of almost the entire cDNA insert of clone pk0010.e4 is shown in SEQ. FROM IDENT. NO 6; the deduced amino acid sequence of this cDNA is shown in SEQ. FROM IDENT. NO: 7. A BLASTX search using the nucleotide sequences established in the SEC. FROM IDENT. NO: 6 results in a pLog value of 132 versus the sequence of Arabidopsis thaliana. The amino acid sequence that is established in SEC. FROM IDENT. NO: 5 is 74.5% similar to the Arabidopsis thaliana sequence, and the amino acid sequence that is established in SEC. FROM IDENT. NO: 7 is 80.3% similar to the sequence of Arabidopsis thaliana. The two Limnanthes sequences are 8d.0% similar to each other, suggesting that both sequences of Limnanthes encode proteins of similar function. The BLAST scores and probabilities indicate that the present nucleic acid fragments code for a portion of the beta-ketoacyl-CoA synthase homologue of Limnanthes douglasii and the entire beta-ketoacyl-CoA synthase homologue of Limnanthes douglasii. However, the oil in Limnanthes is mainly composed of very large chain fatty acids with a delta-5 cis double bond which suggests that the present nucleic acid fragments can indeed encode for acyl-CoA elongase fats instead of beta-ketoacyl-CoA synthases. This is confirmed in example 6, where the expression of Limnanthes elongase in soybean embryos results in an enrichment of fatty acids 20: 0. Therefore, these sequences represent the first sequences of Limnanthes douglasii that code for fatty acyl-CoA elongases.

EXAMPLE 5 Expression of chimeric cells in monocotyledonous cells A chimeric gene comprising a cDNA encoding an enzyme involved in lipid biosynthesis can be constructed in direct orientation with respect to the 27 kD zein promoter of maize that is located 5 'to the cDNA fragment, and the 3 rd end. of 10 kD zein that is located 3 'to the cDNA fragment. The cDNA fragment of this gene can be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or SmaI) can be incorporated into the oligonucleotides to provide adequate orientation of the DNA fragment when inserted into the digested vector pML103, as described below. Then the amplification of this is done in standard PCR. The amplified DNA is then digested with Ncol and Smal restriction enzymes, and fractionated on an agarose gel. The appropriate band can be isolated from the gel and can be combined with a 4.9 kb Ncol-Smal fragment of plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest treaty ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209) and presents the accession number ATCC 97366. The DNA segment from pML103 contains a 10.5 kb Sall-Ncol promoter fragment of the 27 kD zein gene from maize and a 0.96 kb Smal-SalI fragment from the 3 'end of the 10 kD zein gene of the maize in the vector pGem9Zf (+) (Promega) The vector and the inserted DNA can be ligated at 15 ° C during the night, essentially as described (Maniatis). The ligated DNA can then be used to transform E. coli XLl-Blue (Epicurain Coli XL-1 Bluemr; Stratagene). Bacterial transformants can be analyzed by restriction enzyme digestion of the plasmid DNA and limited analysis of the nucleotide sequence using the dideoxy chain termination method (Sequenase * DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct may comprise a chimeric gene encoding, "in the 5 'to 3' direction, the 27 kD corn zein promoter, a cDNA fragment encoding an enzyme involved in lipid biosynthesis and the 3 'region of 10 kD zein The chimeric gene described above can be introduced into the maize cells by the following procedure: Immature maize embryos of the developing caryopses can be dissected from crosses of inbred corn lines H99 and LH132 Embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long Embryos are then placed with the shaft side facing down, and in contact with N6 medium solidified in agarose (Chu) et al., (1975) Sci. Sin. Peking 18: 659-668.) The embryos are kept in the dark at 27 ° C. Friable embryogenic calli consist of undifferentiated masses of cells with somatic proembroids. cos and embroids born on suspension structures that proliferate from the scutellum of these immature embryos. The embryogenic calli isolated from the primary explant can be cultured in N6 medium and subcultured in this medium every 2 or 3 weeks. Plasmid p35S / Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt Germany) can be used in the transformation experiments in order to provide a selectable marker. This plasmid contains the Pat gene (see European patent publication 0 242 236) which codes for phosphinotrisine acetyltransferase (PAT). The PAT enzyme confers resistance to the herbicidal glutamine synthase inhibitors such as phosphinotrisine. The pat gene in p35S / Ac is under the control of the 35S promoter of cauliflower mosaic virus (Odell et al (1985) Nature 313: 810-612) and the 3 'region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The particle bombardment method (Klein T. M. et al. (1987) Nature (London) 327: 70-73) can be used to transfer genes to callus culture cells. According to this method, gold particles (1 ml in diameter) are coated with DNA using the following technique. 10 μg of plasmid DNA is added to 50 μl of a suspension of gold particles (60 mg per ml). Calcium chloride (50 μl of a 2.5 M solution) and spermidine free base (20 μl of a 1.0 M solution) are added to the particles. The suspension is swirled during the addition of these solutions. After 10 minutes, the tubes are centrifuged briefly (5 sec at 15,000 rpm) and the supernatant is removed. The particles are resuspended in 200 μl of absolute ethanol, centrifuged again and the supernatant is removed. Ethanol rinsing is carried out again and the particles are resuspended in a final volume of 30 μl of ethanol. A 5 μl aliquot of the gold particles coated with DNA can be placed in the center of a Kapton flight disc "(Bio-Rads Labs) The particles are then accelerated into the tissue of corn with a Biolistic equipment" 1 * PDS-1000 / He (Bio Rad Instruments, Hercules CA) using a helium pressure of 6895 kPa (1000 psi), a separation distance of 0.5 cm and a flight distance of 1.0 cm. For the bombardment, the embryogenic tissue is placed on a filter paper on a solidified N6 agarose medium. The tissue is arranged as a thin layer and covered with a circular area approximately 5 cm in diameter.

The Petri layer containing the fabric can be placed in the PDS-1000 / He chamber approximately d cm from the stop screen. The air is then evacuated from the chamber to a vacuum of 711 mm (2 in.) Of mercury. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that explodes when the He pressure in the shock tube reaches 6895 kPa (1000 psi). Seven days after the bombardment, the tissue can be transferred to N6 medium containing glufosinate (2 mg per liter) and lacking casein or proline. The tissue continues to grow slowly in this medium. After an additional 2 weeks, the tissue can be transferred to fresh N6 medium containing glufosinate. After 6 weeks, areas of approximately 1 cm in diameter of callus with active growth can be identified in some of the plates containing medium supplemented with glufosinate. These calluses can continue to grow when subcultured in the selective medium. The plants can be regenerated from transgenic callus by first transferring tissue groups of N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks, the tissue can be transferred to the regeneration medium (Fromm et al., (1990) Bio / Technology S: 833-d39).

EXAMPLE 6 Expression of chimeric genes in dicotyledonous cells A seed-specific expression cassette consisting of the promoter and transcription terminator of the gene coding for the β subunit of the phaseolin storage protein can be used from Phaseolus vulgaris bean (Doyle, JJ et al (1986) J. Biol Chem. 261: 9228-9238) for the expression of the present enzymes involved in lipid biosynthesis, in transformed dicots. The phaseolin cassette includes approximately 500 nucleotides towards the 5 'end (5!) From the translation start codon and approximately 1650 nucleotides towards the 3' end (3 ') from the phaseolin translation detection codon. Between the 5 'and 3' regions are unique restriction endonuclease sites (which include the translation start codon ATG), Smal, Kpnl and Xbal. The entire cassette is flanked by HindIII sites. A cDNA fragment of this gene can be generated by polymerase chain reaction (PCR) of the clone cDNA using the appropriate oligonucleotide primers. Cloning sites can be incorporated within the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. The amplification is then carried out as described above, and the isolated fragment is inserted into a vector pUC18 which transports the seed expression cassette. Then the dicotyledonous embryos can be transformed with the expression vector comprising sequences encoding enzymes involved in lipid biosynthesis. To induce somatic embryos, cotyledons 3-5 mm long can be grown dissected from the sterilized surface, from immature seeds of the chosen dicotyledons, in light or dark at 26 ° C, on an appropriate agar medium for 6 hours. -10 weeks. Somatic embryos which produce secondary embryos are then removed and placed in a suitable liquid medium. After repeated selection by groups of somatic embryos which multiply so early, embryos in globular stages, and suspensions are maintained as described below. The dicotyledonous embryogenic suspension cultures can be maintained in a liquid medium of 35 ml on a rotary shaker, 150 rpm at 26 ° C with fluorescent lights in a protocol of 16: 8 hours of day / night. The cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium. The dicotyledonous embryogenic suspension cultures can then be transformed by the particle bombardment method (Klein T.M. et al. (1987) Nature (London) 327: 70-73, U.S. Patent No. 4,945,050). A DuPont Biolistic instrument "5- PDS1000 / HE (retro-placement helium) can be used for these transformations." A selectable marker gene which can be used to facilitate the transformation of plants is a chimeric gene constituted by the 35S promoter of the mosaic virus. of cauliflower (Odell et al., (1985) Nature 313. - 810-812), the hygromycin phosphotransferase gene of plasmid pJR225 (from E. coli; Gritz L. et al (1983) Gene 25: 179-188) and the 3 'region of the nopaline synthase gene of the T-DNA of the Ti plasmid of Agrobacterium turne f hundreds. The seed expression cassette comprising the 5 'region of phaseolin, the fragment coding for the enzyme involved in lipid biosynthesis and the 3' region of phaseolin can be isolated as a restriction fragment. This fragment can then be inserted into a restriction site unique to the vector presenting the marker gene. To 50 μl of a suspension of 1 μm gold particles in 60 mg / ml are added (in order): 5 μl of DNA (1 μg / μl), 20 μl of spermidine (0.1 M) and 50 μl of 2.5 M CaCl2. The particle preparation is then stirred for 3 minutes, centrifuged in a microcentrifuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μl of 70% ethanol and resuspended in 40 μl of anhydrous ethanol. The DNA / particle suspension can be sonicated three times for one second at a time. Then 5 μl of the gold particles coated with DNA are loaded into each macrocarrier disk. Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60 x 15 mm petri dish, and the residual liquid is removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 tissue plates are usually bombarded. The membrane rupture pressure is set at 7584 kPa (1100 psi), and the chamber evacuated to a vacuum of 711 mm (28 inches) of mercury. The fabric is placed approximately 8.9 cm (3.5 inches) away from the retention mesh and is bombarded three times. After the bombardment, the weave can be divided in half and put back into liquid and cultivated as described above. Five to seven days after the bombardment, the liquid medium can be exchanged with fresh medium, and eleven to twelve days after the bombardment can be replaced with fresh medium containing 50 mg / ml of hygromycin. This selective medium can be refreshed every week. Seven to eight weeks after the bombardment can be seen transformed green tissue that grows from non-transformed necrotic embryo groups. The isolated green tissue is removed and inoculated in individual flasks to generate cultures of transformed embryogenic suspension, propagated in a clonal and new manner. Each new line can be treated as a phenomenon of independent transformation. These suspensions can then be subcultured and maintained as groups of immature embryos or can be regenerated into whole plants by maturation and germination of the individual somatic embryos.

Expression of ta-5-acyl-CoA desaturase and acyl-CoA elongase fat from Limnanthes in soybean embryos To confirm the identity and activity of the nucleic acid fragments that are established in the SEC. FROM IDENT. NO.-l (which codes for the delta-5-acyl-CoA lipid desaturase from Limnanthes) and SEC. FROM IDENT. NO: 4 (encoding Limnanthes acyl-CoA elongase fat), these nucleic acids are cloned individually into an expression vector in vivo. The DNA inserts in the pcDNAII library cloning vector are flanked by Notl sites which allows the removal of the entire cDNA insert by digestion with Notl. The plasmids coding for delta-5-acyl-CoA desaturase and the acyl-CoA elongase fat are digested with Notl, the cDNA fragment is isolated, purified and ligated into the pKS67 vector (described below) following standard biology techniques molecular.

A pZBLlOO plasmid is constructed, which contains chimeric genes to allow the expression of hygromycin B phosphotransferase in certain bacteria and in plant cells, from the genetic elements: a) T7 promoter + Shine-Delgarno sequence / hygromycin B phosphotransferase (HPT) / terminator T7, b) 35S promoter of cauliflower mosaic virus (CaMV) / hygromycin B phosphotransferase (HPT) / nopaline synthase (NOS3 'from T-DNA of Agrobacterium turn faciens, and c) plasmid vector pSP72 (Promega) with the coding region of b-lactamase (ampicillin resistance gene) removed. The HPT gene is amplified by PCR from E. coli strain W677, which contains a pJR225 plasmid derived from Klebsiella. Starting with the pSP72 vector, the elements are assembled into a single plasmid using standard cloning methods (Maniatis). The pZBLlOO plasmid therefore contains in T7 promoter / HPT / T7 terminator cassette for the expression of the HPT enzyme in certain strains of E. coli, such as NovaBlue (DE3) (Novagen), are lysogenic for lambda DE3 (which presents the T7 RNA polymerase gene under the lacUV5 control). Plasmid pZBL 100 also contains the 35S / HPT / NOS cassette for constitutive expression of the HPT enzyme in plants, such as soybeans. These two expression systems allow selection for growth in the presence of hygromycin which can be used as a means to identify cells that contain the plasmid in both bacterial and plant systems. Plasmid pZBLlOO also contains three unique restriction endonuclease sites suitable for the cloning of other chimeric genes within this vector. Plasmid pCW109 is derived from a commercially available plasmid pUC18 (Gibco-BRL) by inserting it into the HindIII site of the cloning vector pUC18 with a 5 'non-coding region of 555 bp, (containing the promoter region) of the b-conglycinin gene followed by the multiple cloning sequence containing the restriction endonuclease sites for Ncol, Smal, Kpnl and Xbal and then 1174 bp of the 3'-untranslated region of phaseolin of common bean within the HindIII site. The promoter region of b-conglycinin used is an allele of the published b-conglycinin gene (Doyle et al. (1986) J. Biol. Chem. 261: 9228-9238) due to differences in 27 nucleotide positions. A unique Notl site is introduced into the cloning region between the conglycinin promoter and the 3 'end of phaseolin in pCW109 or cobal digestion and Xbal followed by removal of the ends of single-stranded DNA with mung bean exonuclease. . Linkers Notl (New England Biochemical catalog number NEB 1125) are ligated into the linearized plasmid to produce the plasmid pAW35.

Plasmid pML18 consists of the constitutive and non-specific promoter of tissue, of cauliflower mosaic virus (35S) (Odell, JT et al, (1985) Na ture 313: 810-812; Hull et al (19d7) Virology 86: 432-493), which activates the expression of the gene for neomycin phosphotransferase described in (Beck, E. et al (19d2) Gene 15: 327-336) followed by the 3 'end of the nopaline synthase gene including nucleotides d4d to 1550 described by (Depicker et al., (1982) J. "Appl. Genet . 1: 561-574). This transcriptional unit is inserted into the commercial cloning vector pGEM9Z (Gibco-BRL) and is flanked at the 5 'end of the 35S promoter by restriction sites Sil, Xbal, BamHI and Smal, in that order. An additional Sali site is present at the 3 'end of the 3' NOS sequence, and the Xbal, BamHI and Salí sites are unique. The unique Notl site in pML18 is destroyed by digestion with Notl, filling with single chain ends with dNTP and a Klenow fragment followed by religation of the linearized plasmid. Modified pML18 is then digested with HindIII and treated with bovine intestinal phosphatase. The expression cassette of b-conglycinin: Notl: phaseolin in pAW35 is removed by digestion with HindIII and the 1.8 kB fragment is isolated by agarose gel electrophoresis and ligated into the modified and linearized pML18 construct described above. A clone with the desired orientation is identified by digestion with Notl and XbaI to release a 1.08 kb fragment which indicates that the orientation of the conglycinin transcription unit is the same as that of the transcription unit of the selectable marker. The resulting plasmid is named pBS19. The pKS67 vector is prepared by isolating the fragment containing b-conglycinin from pBS19 by digestion with HindIII, isolation by gel electrophoresis and ligation within pZBLlOO digested with HindIII, which has been treated with bovine alkaline phosphatase. Soy bean embryogenic suspension cultures are transformed with the expression vectors by the particle gun bombardment method (Klein, TM et al. (1987) Na ture (London) 327: 70-73, US Patent No. 4,945,050 ). The maintenance of the transgenic embryos, the preparation of oils and the measurement of the fatty acid content by gas chromatography is carried out as indicated in PCT publication W093 / 11245 (incorporated herein by reference).

Demonstration of elongasa activity The percent of 16: 0 fatty acid accumulation decreases in soybean embryos expressing the acyl-CoA elongasa fat, while the levels of fatty acids 20: 0 are markedly increased. Figure 3 represents a TABLE 4 Percent distribution of fatty acid in transgenic embryos of soybean expressing acyl-CoA elongase fat from Limnanthes Embryo 16: 0 20: 0 4155 wild type 12.9 0.2 323 wild type 13.0 0.4 312 wild type 18.4 0.6 4111 wild type 15.4 0.6 4102 wild type 15.2 0.6 195 wild type 15.2 0.6 transgenic 129 7.4 9.3 161 transgenic 7.6 8.2 163 transgenic 7.9 7.7 175 transgenic 8.4 11.0 2211 transgenic 8.5 7.9 341 transgenic 6.4 10.8 Figure 4 shows the linear relationship between the decrease in 16: 0 fatty acid content and increase in the 20: 0 fatty acid content in the expression in -6l - chromatographic analysis of oils derived from soybean embryos from wild-type non-transgenic (Figure 3 (A)) and from transgenic soybean embryos expressing the Limnanthes acyl-CoA elongase fat (Figure 3 (B)). The peaks corresponding to the different fatty acids present are indicated. The amount of 16: 0 and 18: 2 fatty acids decrease in the oils of the soybean embryos expressing acyl-CoA elongase fat, when compared to the oil derived from non-transgenic wild-type soybean embryos. further, the amount of 20: 0 fatty acids is greatly enriched in the oils of the transgenic embryos. Table 4 shows the quantified distribution of 16: 0 and 20: 0 fatty acids in wild-type soybean embryos and soybean embryos expressing Limnanthes acyl-CoA elongase fat. The 20: 0 fatty acid levels in wild type embryos vary from 0.2 to 0.6%, while the embryos that express acyl-CoA elongase fat, the 20: 0 fatty acid percent, varies from 7.7% to 11.0%. - 6Í - Transgenic soybean embryos from the acyl-CoA elongasa fat from Limnanthes.

Demonstration of the activity of ta-5 desaturase Transgenic soybean embryos that express delta-5-acyl-CoA desaturase from Limnanthes produce 16: 1 fatty acids that are not seen in wild-type embryos. The fatty acid distribution in soybean embryos expressing delta-5 desaturase is illustrated in Figure 5, which shows the chromatograms that correspond to oils derived from wild-type soybean embryos (Figure 5 (A )) and soybean embryos expressing delta-5 acyl-CoA desaturase from Limnanthes (Figure 5 (B)). Table 5 shows the quantified percent distribution of 16: 0, 16: 1 delta-5, 18: 0 and 18: 1 delta-5 in wild type embryos and transgenic embryos expressing delta-5 acyl-CoA desaturase Limnanthes: TABLE 5 Percent distribution of fatty acid in transgenic embryos of soybeans expressing delta-5 acyl-CoA desaturase from Limnanthes embryo 16: 0 16: 1? 5 18: 0 18: 1? 5 216. 0 (wild type) 13.03052 0 2.47064 0 216.1 (wild type) 10.60185 0 1.76857 0 216.2 (wild type) 11.95366 0 1.67544 0 218.0 (wild type) 12.93328 0 2.15752 0 218.5 (wild type) 11.57688 0 2.24243 0 220.0 (transgenic) 10.17740 3.63283 1.25350 0.57574 220. 1 (transgenic) 8.99496 4.27946 1.22194 0.71544 220.2 (transgenic) 9.78203 2.86631 1.57083 nd 220. 3 (transgenic) 9.47315 3.35682 1.49796 0.60828 220. 4 (transgenic) 12.16690 2.46238 1.84877 0.45737 220. 5 (transgenic) 12.22757 2.75365 2.38873 0.53878 220. 6 (transgenic) 11.72778 2.43411 2.37860 0.57778 220.7 (transgenic) 9.31376 3.39302 1.33830 0.59855 220. 8 (transgenic) 9.48067 3.66554 1.45045 0.66508 220. 9 (transgenic) 9.37735 3.47590 0.95774 0.75598 217. 1 (transgenic) 9.86364 3.56592 1.51745 0.64637 217. 2 (transgenic) 11.03674 2.79068 1.92739 0.55140 217.3 (transgenic) 13.57543 2.45928 2.26611 0.56306 217.4 (transgenic) 11.33959 2.88931 1.73524 0.53747 217. 5 (transgenic) 9.61358 3.40842 1.94986 0.73811 217. 6 (transgenic) 10,54626 3.11490 1.69327 nd 217.7 (transgenic) 11,60064 3.34681 2.77254 0.78978 217. 8 (transgenic) 13.76804 1.41011 3.41123 nd 217.9 (transgenic) 11.21888 2.98101 1.87345 0.61650 nd = not enough delta-5 is produced to be integrated by the instrument.

To confirm the position of the double bond catalyzed by the desaturase, the positions of the double bonds of monounsaturated fatty acids are established by GC analysis for disulfide derivatives of fatty acid methyl esters, as described by Yamamoto, K. et al. . (1991) Chem Phys Lipids 60: 39-50 and as illustrated in Figure 6. Fatty acid methyl esters prepared from soybean embryos expressing delta-5 acyl-CoA desaturase from Limnanthes are reacted with disulfide of dimethyl as previously described (Yamamoto, K. et al. (1991) Chem. Phys. Lipids 60: 39-50). This reaction converts the double bonds of the methyl esters of unsaturated fatty acids to dimethyl disulfide adduct (DMDS). When analyzed by GC-MS, these derivatives provide ions that are diagnostic for the positions of the double bonds in - 6β - the fatty acids. The DMDS derivatives of the fatty acid methyl esters of the transgenic soybean embryos are analyzed by GC-MS. These derivatives are separated using a column of 0.25 mm (inner diameter x 30 m of HP-INNOWax (Hewlett Packard), with an oven temperature of a gas chromatograph temperature HP6890 programmed for ld5 ° C (5 minutes retention) to 237 ° C (25 minutes of retention) at a speed of 7.5 ° C / min The mass spectrum of the separated DMDS derivatives is obtained, using a selective mass detector HP5973 that is placed in interface with the gas chromatograph. The DMDS derivatives of methylhexadecenoic acid (16: 1) are identified using a selected ion scanner for 362 m / z, which corresponds to the molecular ion of the DMDS derivatives of methyl 16: 1? This results in an identification of two peaks with retention times between 18.5 and 19.5 minutes (Figure 6 (A)). The mass spectrum of the largest of these peaks contains abundant ions with m / z of 161 and 201 (Figure 6 (B)). The masses of these ions are consistent with the presence of the double bond at the delta-5 carbon atom. The ion 161 m / z (fragment Y) is the mass expected for the carboxyl portion of the DMDS derivative of methyl 16: l? 5 and the ion of 201 m / z (fragment X) is the mass expected for the methyl end of the DMDS derivative 16: l? 5. It is also consistent with the identification of this peak as a derivative of 16.-1? 5 DMDS in the 129 m / z ion, which is generated by rearrangement of the fragment and with the loss of 32 m / z. In general, the Y-32 ion is considered as a diagnostic ion for the DMDS derivatives of methyl esters of monounsaturated fatty acids (Francis, G.W. (1981) Chem. Phys. Lipids 29: 369-374). It is noteworthy that the second, smaller peak in Figure 6 (A) is identified as the DMDS derivative of methyl 16: l? 9 (results not shown). This fatty acid, in contrast to 16.-1? 5, is detectable in small amounts in virtually all plant tissues. DMDS derivatives of methyloctadecenoic acid (18: 1) are initially identified using a selected ion scan for 390 m / z (Figure 6 (C)), which corresponds to the mass of the molecular ion of these adducts. As shown in Figure 6 (D), fragmentation of the DMDS derivative of methyl 18: l? 5 can be expected to generate ions of 161 m / z, 229 m / z and 129 m / z corresponding to the Y fragments. , X and Y-32, respectively. These ions are detected in a projection in the front of the peak that corresponds to the methyl derivative 18: l? 9, the main monounsaturated fatty acid of the soybean embryos. The results presented in this document establish the presentation of 16: l? 5 and 18: l? 5 in methyl esters of fatty acid derived from transgenic embryos of soybeans that express delta-5-acyl-CoA desaturase * - 6th-of Limnanthes. None of the fatty acids is detectable in derivatives prepared from wild-type soybean embryos. These experiments show that when expressed in soy bean embryos, the acyl-CoA elongase Limnanthes fat (SEQ ID NO: 5) catalyzes the production of arachidonate (20: 0) from palmitate (16: 0). ). These experiments also show that Limnanthes acyl-CoA desaturase (SEQ ID NO: 2) codes for a delta-5 desaturase which produces 16: 1 delta-5 and 18: 1 delta-5 fatty acids when expressed in transgenic embryos of soybean. These experiments are the first demonstration of the activity of delta-5-acyl-CoA desaturase and the acyl-CoA elongase fat, from Limnanthes douglasii, whose sequences are established in SEC. FROM IDENT. NO: 2, SEC. FROM IDENT. NO: 5 and SEC. FROM IDENT. NO: 7. The expression of the acyl-CoA elongase Limnanthes fat in other crops that produce oil will increase the amounts of C20: 0 from about less than 1% to more than about 15%. The expression of the acyl-CoA elongase fat of Limnanthes and the delta-5-acyl-CoA desaturase in other harvests of oilseeds will have the result of producing oils 20: 1 delta-5 which can then be used in the production of compounds industrially useful.

EXAMPLE 7 Expression of chimeric genes in microbial cells The cDNAs encoding the present enzymes involved in lipid biosynthesis can be inserted into the E. coli T7 expression vector, pBT430. This vector is a derivative of pET-3a (Rosenberg et al (1987) Gene 56: 125-135) which uses the T7 RNA polymerase system of bacteriophage / T7 promoter system. Plasmid pBT430 is constructed by first destroying the EcoRI and HindIII sites in pET-3a at their original positions. An oligonucleotide adapter containing the EcoRI and HindIII sites is inserted into the BamHI site of pET-3a. This generates pET-3aM with "additional unique cloning sites for insertion of the genes into the expression vector." Then, the Ndel site at the translation start position is converted to an Ncol site using oligonucleotide-directed mutagenesis. PET-3aM DNA in this region, 5'-CATATGG is converted to 5'-CCCATGG in pBT430. The plasmid DNA containing cDNA can be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment can then be purified on a low melting agar gel (FMC) NuSieve GTGm 1%. Shock absorber and agarose containing 10 μg / ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase * (Epicenter Technologies) according to the manufacturer's instructions, precipitated with ethanol, dried and resuspended in 20 μl of water. Appropriate oligonucleotide adapters can be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA). The fragment containing the ligated adapters can be purified from the excess of adapters using agarose with low melting point, as described above. The pBT430 vector is targeted, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol / chloroform as described above. The prepared vector pBT430 and the fragment can then be ligated at 16 ° C for 15 hours, followed by transformation into electrocompetent DH5 cells (GIBCO BRL). Transformants can be selected on agar plates containing LB medium and 100 μg / ml ampicillin. Transformants containing the gene encoding the enzyme involved in lipid biosynthesis are then analyzed to determine the correct orientation with respect to the T7 promoter by restriction enzyme analysis. For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J. "Mol. Biol. 185: 113-130) The cultures are grown in LB medium containing 100 mg / L of ampicillin at 25 ° C. At an optical density of 600 nm of about 1, IPTG (isopropylthio-β-galactoside, the inducer) at a final concentration of 0.4 mM and the incubation can be continued for 3 h at 25 ° C. The cells are then harvested by centrifugation and resuspended in 50 μl of 50 mM Tris-HCl at pH 8.0 containing DTT. 0.1 mM and 0.2 mM phenyl methyl sulfonyl fluoride A small amount of 1 mM glass spheres can be added and the mixture is sonicated three times for approximately 5 seconds each time with a microprobe sonicator. and the protein concentration in the supernatant is determined You can separate 1 μg of the protein from the soluble solution of the culture by SDS-polyacrylamide agarose gel electrophoresis. The gels can be observed to determine - the protein bands that migrate at the expected molecular weight. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (34)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated fragment of nucleic acid encoding all or a substantial portion of a delta-5-acyl-CoA desaturase, characterized in that it comprises a member selected from the group consisting of: (a) an isolated fragment of nucleic acid that codes for all or a substantial portion of the amino acid sequence set forth in SEC. FROM IDENT. N0: 2; (b) an isolated fragment of nucleic acid that is substantially similar to an isolated fragment of nucleic acid encoding all or a portion • Substantial amino acid sequence established in SEC. FROM IDENT. N0: 2; and (c) an isolated fragment of nucleic acid, which is complementary to (a) or (b).
2. 2. The isolated fragment of nucleic acid, according to claim 1, characterized in that the nucleotide sequence of the fragment comprises all or a portion of the sequence established in SEQ. FROM IDENT. N0: 1 3. A chimeric gene, characterized in that it comprises the nucleic acid fragment according to claim 1, operably linked to suitable regulatory sequences. 4. A transformed host cell, characterized in that it comprises the chimeric gene according to claim 3. 5. A delta-5-acyl-CoA desaturase polypeptide, characterized in that it comprises all or a substantial portion of the amino acid sequence that is established in the SEC. FROM IDENT. N0: 2 6. An isolated fragment of nucleic acid, which codes for all or a substantial portion of a fatty acyl-CoA elongase, characterized in that it comprises a member selected from the group consisting of: (a) an isolated fragment of nucleic acid which encodes for all or a substantial portion of the amino acid sequence that is established in a member that is selected from the group consisting of SEQ. FROM IDENT. NO: 5 and SEC. FROM IDENT. N0: 7; (b) an isolated fragment of nucleic acid that is substantially similar to an isolated fragment of nucleic acid encoding all or a substantial portion of the amino acid sequence that is established in a member selected from the group consisting of the SEC . FROM IDENT. NO: 5 and the SEC. FROM IDENT. NO: 7; and (c) an isolated fragment of nucleic acid that is complementary to (a) or (b). 7. The isolated fragment of nucleic acid, according to claim 6, characterized in that the nucleotide sequence of the fragment comprises all or a portion of the sequence that is established in a member • which is selected from the group consisting of the SEC. FROM IDENT. NO: 4 and SEC. FROM IDENT. NO: 6. 8. A chimeric gene, characterized in that it comprises the nucleic acid fragment according to claim 6, operably linked to suitable regulatory sequences. 9. A transformed host cell, characterized in that it comprises the chimeric gene according to claim 8. 10. An acyl-CoA elongase fat polypeptide, characterized in that it comprises all or a substantial portion of the amino acid sequence that is established in a member which is selected from the group consisting of the • SEC. 'DE IDENT. NO: 5 and SEC. FROM IDENT. NO: 7 11. A method for altering the level of expression of an enzyme involved in the biosynthesis of lipids in a host cell, characterized in that it comprises: (a) transforming a host cell with the chimeric gene according to any of claims 3 and 8; and (b) growing the transformed host cell produced in step (a) under conditions that are suitable for the expression of the chimeric gene, wherein the expression of the chimeric gene results in the production of altered levels of an enzyme involved in biosynthesis of lipids in the transformed host cell. 12. A method for producing a desaturated fatty acid comprising a double bond at the delta-5 position in a host cell, the method is characterized in that it comprises: (a) transforming a host cell with the chimeric gene according to claim 3, and (b) growing the host cells transformed in the step (a) under conditions which are suitable for the expression of the chimeric gene, wherein the expression of the chimeric gene results in the production of a desaturated fatty acid comprising a double bond at the delta-5 position. 13. A seed, characterized in that it is obtained from an oilseed crop in which the seed comprises a desaturated fatty acid, wherein the fatty acid comprises a double bond at the delta-5 position. 14. The seed according to claim 13, characterized in that the oilseed crop is soybeans. 15. An oil, characterized in that it is obtained from the seed of an oilseed crop, wherein the oil comprises a desaturated fatty acid, wherein the fatty acid comprises a double bond at the delta-5 position. according to claim 15, characterized in that the oilseed crop is soybeans. 17. A method for producing seed oil comprising a desaturated fatty acid, wherein the fatty acid comprises a double bond at the delta-5 position, the method is characterized in that it comprises: (a) transforming a plant cell with the chimeric gene according to claim 3; (b) growing a fertile plant from the transformed plant cell of step (a); (c) obtaining a seed of the plant of step (b); and (d) processing the seed of step (c) to obtain oil, wherein the oil comprises a desaturated fatty acid in which the fatty acid comprises a double bond at the delta-5 position. 18. The method according to claim 17, characterized in that the plant cell is derived from an oilseed crop. 19. The method according to claim 18, characterized in that the oilseed crop is soybeans. 20. The seed oil, characterized in that it is produced by the method according to claim 17. 21. A method for reducing the level of 16 carbon fatty acids in a host cell, characterized in that it comprises: (a) transforming a cell host with the chimeric gene according to claim 8; and (b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene wherein the expression of the chimeric gene results in the reduction of the 16-carbon fatty acid level in the host cell transformed when compared to the level of fatty acids of 16 carbons in a host cell that has not been transformed with the chimeric gene according to claim 8. 22. A method for producing seed oil with reduced levels of carbon fatty acids 16, the method is characterized in that it comprises: (a) transforming a plant cell with the chimeric gene according to claim 8; (b) growing a fertile plant from the transformed plant cell of step (a); (c) obtaining a seed of the plant of step (b); and (d) processing the seed of step (c) to obtain oil, wherein the oil comprises a lower level of fatty acids of 16 carbons in comparison with the oil obtained from a seed obtained from a plant that has grown from a plant cell that has not been transformed with the chimeric gene according to claim 8. 23. The method according to claim 22, characterized in that the plant is a "oilseed crop" 2. The method according to claim 23, characterized in that the oilseed crop is soybeans 25. The seed oil, characterized in that it is produced by the method according to claim 22 . 26. A method for increasing the level of fatty acids of carbon 20 in a host cell, characterized in that it comprises: (a) transforming a host cell with the chimeric gene according to claim 8; and (b) growing the transformed host cell in step (a) under conditions that are suitable for expression of the chimeric gene wherein the expression of the chimeric gene results in an increase in the level of fatty acids of 20 carbons in the host cell transformed when compared to the level of fatty acids of 20 carbons in a host cell that has not been transformed with the chimeric gene according to claim 8. 27. A method for producing seed oil with increased levels of fatty acids of 20 carbons, characterized in that it comprises: (e) transforming a plant cell with the chimeric gene according to claim 8; (f) growing a fertile plant from the transformed plant cell of step (a); (g) obtaining a seed of the plant of step (b); and (h) processing the seed of step (c) to obtain an oil wherein the oil comprises a higher level of fatty acids at 20 carbons compared to the oil obtained from a seed obtained from a plant that has grown to Starting from a plant cell that has not been transformed with the chimeric gene according to claim 8. 28. The method according to claim 27, characterized in that the plant is an oilseed crop. 29. The method according to claim 28, characterized in that the oilseed crop is soybeans. 30. Seed oil, characterized in that it • is produced by the method in accordance with the claim 31. A method for obtaining a nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding an enzyme involved in lipid biosynthesis, the method is characterized in that it comprises: (a) probing a cDNA or genomic library with the nucleic acid fragment according to any of claims 1 and 6; (b) identifying a cDNA clone that hybridizes to the nucleic acid fragment according to any of claims 1 and 6; (c) isolating the DNA clone identified in step (b); and (d) sequencing the cDNA or genomic fragment comprising the clone isolated in step (c) wherein the sequenced nucleic acid fragment codes for all or a substantial portion of the amino acid sequence encoding an enzyme involved in the biosynthesis of lipids. 32. A method for obtaining a nucleic acid fragment that encodes a substantial portion of an amino acid sequence that codes for an enzyme involved in lipid biosynthesis, the method is characterized in that it comprises: (a) synthesizing an oligonucleotide primer that corresponds to a portion of the sequence that is established in any of the SECs. FROM IDENT. NO: 1, 4 and 6; and (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector wherein the amplified nucleic acid fragment codes for a substantial portion of a sequence of amino acids that code for an enzyme involved in lipid biosynthesis. 33. The product of the method according to claim 31. 34. The product of the method according to claim 32.