AU5417299A - Method to alter the fatty acid metabolism in plants - Google Patents

Description

WO00/08172 PCTIEP99/05543 Method to Alter the Fatty Acid Metabolism in Plants Description The present invention relates to amino acid and nu cleic acid sequences involved in the fatty acid elongation metabolism of a plant, in particular a maize plant, to vectors containing the nucleic acid sequences, to antibodies directed against the amino acid sequences, to methods to obtain plants trans formed with the nucleic acid sequences of the pre sent invention and to methods of use for the nu cleic acid sequences of the present invention. Seed storage lipids of higher plants are primarily made up of fatty acids containing 16 and 18 carbon atoms. These fatty acids are located in the seed oils of various plant genera. There are only a few plants, such as the Cruciferae, which accumulate oils of C20 and C22, which are referred to as very long chain fatty acids (VLCFAs). The commercial use of vegetable oils depends heavily upon the presence of VLCFAs. Erucic acid (22:1) has detrimental nu tritional effects and is therefore undesirable in edible oils. Wild-type rape seed and many Brassica species, however, contain eicosenoic (20:1) and erucic acids as major components of their seed oils. However, through concerted breeding efforts, canola lines that are almost devoid of erucic acid have been developed. This has been achieved by the WO00/08172 PCT/EP99/05543 -2 introduction of recessive alleles at two loci that control the elongation of C18 fatty acids. On the other hand, for industrial uses, vegetable oils with a high erucic acid level have been proven to be useful. These oils can be used as diesel fuel and as a raw material for an array of products, such as plastics, pharmaceuticals and lubricants. It is known that in plants de novo fatty acid syn thesis (FAS) is localised in the plastids and in volves intermediates bound to acyl carrier proteins (ACPs). The FAS system is believed not to produce fatty acids with chain lengths of more than 18 car bon atoms. The products of the plastid FAS are ex ported and converted to acyl-coenzyme A (acyl-CoA) derivatives that are thought to serve as substrates for a microsomal fatty acid elongation (FAE) sys tem. The FAE is membrane associated and its nature and mechanism of action are partly unknown. It is believed that FAE in plants involves a four-step mechanism similar to FAS, except that CoA, rather than ACP, is the acyl carrier. Four different reac tions appear to be involved in the elongation sys tem of plants, which are (1) condensation of 18:1 CoA with malonyl-CoA to form carbon dioxide and a 9-ketoacyl-CoA in which the acyl moiety has been elongated by two carbons. Subsequent reactions are the reduction to 9-hydroxyacyl-COA, dehydration to an enoyl-CoA and a second reduction to yield the elongated acyl-CoA. In summary, the fatty acid car bon chain is elongated from C18 to C22 by the se quential addition of two C2 moieties from malonyl coenzyme A (CoA) to a C18 carbon skeleton.

WO00/08172 PCT/EP99/05543 -3 Through the development of plant genetic engineer ing techniques, it is possible to transform and re generate plant species to provide plants which have novel and advantageous features. Plant genetic en gineering techniques may be employed in producing insect-resistant plants, herbicide-resistant plants, draught-resistant plants and for instance also plants containing desirable products or plants devoid of undesired naturally occurring substances. Due to the potential commercial value of plants ex hibiting a modified fatty acid elongation system, DNA sequences involved in the FAE system, in par ticular 9-ketoacyl-CoA synthases (KCS) have been cloned from for instance jojoba and Arabidopsis (Lassner et al., The Plant Cell, February 1996 8, 281-292 and James et al., The Plant Cell, March 1995 7, 309-319). The seed oil of jojoba has the intrinsic feature that it consists of waxes rather than the triacylglycerols constituting other seed oils. The waxes are known to be esters of monoun saturated fatty acids and alcohols. Acyl CoA's are precursors of both the fatty acid and the fatty al cohol moieties of the wax esters. More than 90% of these fatty acids and alcohols have chain lengths longer than 18 carbon atoms, indicating the pres ence of an active acyl coenzyme A elongation sys tem. As in rape seed, malonyl CoA and possibly acyl CoA serve as substrates for VLCFA synthesis. The enzyme shown to be involved in the production of the wax esters is a S-ketoacyl coenzyme A synthase involved in the acyl coenzyme A elongation pathway. Furthermore, a cDNA coding for a S-ketoacyl coen zyme A synthase from Arabidopsis was cloned and shown to be involved in the synthesis of very long WO00/08172 PCT/EP99/05543 -4 chain fatty acids in the seed (James, Jnr. et al., The Plant Cell, March 1995, 7, 309-319). However, none of the known and cloned nucleic acid sequences involved in the fatty acyl coenzyme A elongation pathway is derived from a monocotyledo nous plant. Due to the high commercial value of crop plants and differences, for instance with re spect to codon usage between monocotyledonous and dicotyledonous plants, it is desirable to provide nucleic acid sequences useful for cloning genes in volved in the fatty acyl coenzyme A elongation pathway in monocotyledonous plants. The present invention solves this problem by pro viding a purified nucleic acid sequence for use in cloning nucleic sequences encoding a protein with the activity of a fatty acid elongase wherein the nucleic acid sequence comprises a nucleic acid se quence selected from the group consisting of a)a nucleic acid sequence encoding any one of the amino acid sequences identified in SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37 and 39 or a complementary strand or part thereof, b)a nucleic acid sequence which hybridises to the nucleic acid sequence defined in a) or a complementary strand thereof and c)alleles or derivatives of the nucleic acid sequences defined in a) or b).

WO00/08172 PCT/EP99/05543 -5 In a particularly preferred embodiment, the present invention relates to a purified nucleic acid se quence according to the above, which is selected from the group consisting of ai) SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 36 and 38 or a complementary strand or part thereof, bi) a nucleic acid sequence which hybridises to the nucleic acid sequence defined in ai) or a complementary strand thereof, ci) a nucleic acid sequence which is degener ated as a result of the genetic code to the nucleic acid sequence defined in ai) or bi) or a complementary strand or part thereof and di) alleles or derivatives of the sequence de fined in ai), bi) or ci). Thus, the present invention solves the above prob lem by providing a DNA sequence which is in a pre ferred embodiment the sequence depicted in SEQ. ID. No. 32 or 33. The sequence depicted in SEQ. ID. No. 32 is a maize full length nucleic acid sequence en coding a functional protein with the activity of a fatty acid elongase. The complete amino acid se quence of this protein is depicted in SEQ. ID. No. 1. SEQ. ID. No. 33 represents the coding nucleotide sequence of the cloned nucleic acid sequence de picted in SEQ. ID. No. 32, whereby the latter addi tionally contains 5' and 3' non-coding sequences.

WO00/08172 PCT/EP99/05543 -6 These 5' and 3' non-coding flanking sequences are also part of the present invention. The sequences depicted in SEQ. ID. Nos. 2 to 31 and 34 to 39 are partial nucleotide and corresponding amino acid sequences. These sequences are charac terised by being unique to the sequence depicted in SEQ. ID. No. 32. In fact, the sequences depicted in SEQ. ID. Nos. 2 to 31 and 34 to 39 share little, or hardly any, identity with nucleic acid sequences, or amino acid sequences being derived from plants other than maize and encoding a protein with the activity of a fatty acid elongase. Thus, these se quences are maize specific. Thus, the present invention provides a purified nu cleic acid sequence, in particular a DNA and/or RNA sequence, encoding a plant cytoplasmic protein in volved in fatty acyl-CoA metabolism and/or useful for obtaining such a sequence. Of course, the se quence of the present invention is also useful for obtaining non-coding sequences, such as regulatory sequences adjacent to the sequence encoding the protein involved in fatty acyl-CoA metabolism. The nucleic acid sequences of the present invention are advantageous, as they can be used in processes to alter the composition of very long chain wax fatty acid related products such as very long chain fatty acids, in the following abbreviated as VLCFAs and/or wax esters. The nucleic acid sequences of the present invention, in particular the cDNA de rived sequences, are advantageous also insofar as they allow for the production of male sterile plants and plants, for instance ornamental plants, WO00/08172 PCT/EP99/05543 -7 exhibiting a modified leaf structure, in particular modified leaf edges and a modified wax composition and/or distribution in and/or on the leaves, in particular in the cuticular. The present invention allows for the modification of the wax composition of a transgenic plant so as to improve or create insect, fungus, pest, drought or herbicide resistance. The present invention is particularly advantageous since the nucleic acid sequences mentioned above are specific nucleic acid sequences derived from Zea mays, that is corn or maize. Thus, the present invention provides specific maize nucleic acid se quences for use in cloning nucleic acid sequences encoding a maize protein with the activity of a fatty acid elongase. The present invention also provides nucleic acid sequences capable of enhanc ing, directing and/or expressing such a maize de rived protein. Up to now, no nucleic acid sequences derived from a monocotyledonous plant are known ca pable of cloning such a nucleic acid sequence or capable of expressing such a protein. In the context of the present invention, the term "activity of a fatty acid elongase" relates to an activity associated with the plant fatty acyl-CoA metabolism. In particular, the above activity in volves condensation of malonyl-CoA with a long chain acyl-CoA to yield CO 2 and 9-hydroxyacyl-CoA, having two additional C-atoms, reduction to S-hy droxyacyl-CoA, dehydration to enoyl-CoA and/or a further reduction to elongated acyl-CoA. Thus, the WO00/08172 PCT/EP99/05543 -8 above term relates to any one of the above activi ties, in particular to an activity of a protein ac cording to which very long chain fatty acids, that is VLCFAs, are synthesised from long chain fatty acids. Thus, the protein encoded by the above DNA sequences is involved in the production of C>18, in particular, C20 and C22 and more preferred C>27, in particular C27 to C33, most preferably C28 to C30 fatty acids from fatty acids having equal or less than 18 C-atoms, in particular C16 and C18 fatty acids. In the case where fatty acids having more than 27 C-atoms are synthesised, these may be syn thesised from fatty acids having more than 18 C atoms, for instance C24 fatty acids. In particular, the protein encoded by the above nucleic acid se quences is a condensing enzyme that extends or elongates the chain length of long chain fatty ac ids to very long chain fatty acids. The protein en coded by the nucleic acid sequences of the present invention may, of course, also be part of an acyl CoA elongase complex, for instance a regulatory and/or catalysing element. As explained above, the catalysing function may be a 9-ketoacyl-CoA synthase activity. However, the protein of the pre sent invention may also have 9-ketoacyl-CoA reductase, 9-hydroxyacyl-CoA-dehydratase and/or enoyl-CoA-reductase activity. The protein of the present invention may also function as a fatty acyl-CoA: fatty alcohol O-acyltransforane, i.e. as a wax synthase, forming a wax ester from a fatty alcohol and a fatty acyl residue. The term "a pro tein with the activity of a fatty acid elongase" encompasses any one of the above described activi ties, either alone or in combination.

WO00/08172 PCT/EP99/05543 -9 The nucleic acid sequences of the present invention may be synthetic DNA, genomic DNA or cDNA se quences. Of course, the nucleic acid sequences of the present invention also comprise RNA sequences, for instance mRNA. In addition to the sequences given in SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 36 or 38, the nucleic acid sequences of the present invention may contain further sequences such as regulatory elements necessary for transcription, translation, recombination or integration. These elements may already be used in addition to regulatory elements contained in the nucleic acid sequences of the pre sent invention. Nucleic acid sequences of the present invention in clude those nucleic acid sequences, that is DNA or RNA sequences, which hybridise to the specifically disclosed nucleic acid sequences of SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 36 or 38. Most preferably, these sequences encode a protein having the biological activity of a fatty acid elongase, in particular the maize fatty acid elongase. However, the present invention also relates to regulatory nucleic acid sequences found 5' or 3' to the coding sequences or even regulatory sequences found between the coding sequences of a genomic DNA, that means intron se quences or sequences contained in introns. The nu cleic acid sequences of the present invention may, in a particular preferred embodiment, comprise se quences encoding signal or leader peptides being part of a precursor protein but not being present in the mature protein.

WO 00/08172 PCT/EP99/05543 -10 In the context of the present invention, nucleic acid sequences which hybridise to the specifically disclosed nucleic acid sequences of SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 36 or 38 are sequences which have a degree of 65% to 70% sequence identity to the spe cifically disclosed sequence on nucleotide level. In an even more preferred embodiment of the present invention, nucleic acid sequences which are encom passed by the present invention are sequences which have a degree of identity of more than 70%, 80% or 85% and even more preferred more than 90%, 95% or 99% to the specifically disclosed sequences on nu cleotide level. The present invention also relates to nucleic acid sequences which encode proteins, wherein the amino acid sequence of the proteins have a degree of identity of 75% to 90% on amino acid level, most preferably a degree of identity of more than 90%, 95% or 99% identity on amino acid level to the spe cifically disclosed amino acid sequences of the present invention. Thus, the present invention relates to nucleic acid sequences, in particular DNA sequences which hy bridise under the following conditions to the se quences specifically disclosed: Hybridisation buffer: 1 M NaCl; 1% SDS; 10% dextran sulphate; 100 Ag/ml ssDNA Hybridisation temperature: 650 C First wash: 2 x SSC; 0.5% SDS at room temperature WO 00/08172 PCT/EP99/05543 -11 Second wash: 0.2 x SSC; 0.5% SDS at 650 C. More preferably, the hybridisation conditions are chosen as identified above, except that a hybridi sation temperature and second wash temperature of 680 C, and even more preferred, a hybridisation temperature and second wash temperature of 700 C is applied. The present invention also comprises nucleotide re arrangements, exchanges, substitutions, insertions, deletions or modification of the above mentioned sequences as long as the biological activity of the encoded protein remains essentially the same or is improved. As used herein, the term "promoter" refers to a se quence of DNA, usually upstream (5') to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at the correct site. Promoter sequences are necessary, but not al ways sufficient to drive the expression of the gene. A "promoter fragment" constitutes a fraction of the DNA sequence of the promoter region. A "3' regulatory element" (or "3' end") refers to that portion of a gene comprising a DNA segment, excluding the 5' sequence which drives the initia tion of transcription and the structural portion of the gene, that contains a polyadenylation signal, also called a poly A addition sequence, and any other regulatory signals capable of affecting mes- WO 00/08172 PCT/EP99/05543 -12 senger RNA (mRNA) processing or gene expression. The polyadenylation signal is usually characterised by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. Poly adenylation signals are commonly recognised by the presence of homology to the canonical form 5' AATAA-3', although variations are not uncommon. "Nucleic acid" refers to a large molecule which can be single or double stranded, composed of monomers (nucleotides) containing a sugar, phosphate and ei ther a purine or pyrimidine. The term "nucleotide sequence" refers to a polymer of DNA or RNA which can be single or double stranded, optionally con taining synthetic, non-natural or altered nucleo tide bases capable of incorporation into DNA or RNA polymers. As used herein, "gene" refers to a DNA sequence that codes for a specific protein and its regula tory elements. As used herein, the term "regulatory element" re fers to a nucleotide sequence located upstream (5'), within, and/or downstream (3') to a DNA se quence for a selected gene product whose transcrip tion and/or translation is controlled by said regu latory sequence, potentially in conjunction with the protein biosynthetic apparatus of the cell. "Regulation" or "regulate" refer to the modulation of the gene expression induced by DNA sequence ele ments located primarily, but not exclusively, up stream of (5') the transcription start of the gene. Regulation may result in an all or none response to WO 00/08172 PCT/EP99/05543 -13 a stimulation, or it may result in variations in the level of gene expression. The term "coding sequence" refers to that portion of a gene encoding a protein, polypeptide, or a portion thereof, and excluding the regulatory se quences which drive the initiation of transcrip tion. The coding sequence may be one normally found in the cell, in which case it is called "autologous", or it may be one not normally found in a cellular location, in which case it is termed a "heterologous gene" or "heterologous nucleic acid sequence". A heterologous gene may also be composed of autologous elements arranged in an order and/or orientation not normally found in the cell being transferred with the gene. A heterologous gene may be derived in whole or in part from any source known to the art, including a bacterial genome or episome, eukaryotic nuclear or plasmid DNA, cDNA, or chemically synthesised DNA. The structural gene may constitute an uninterrupted coding region or it may include one or more introns bounded by appro priate splice junctions. The structural gene may be a composite of segments derived from different sources, naturally occurring or synthetic. The term "vector" refers to a recombinant DNA con struct which may be a plasmid, virus, or autono mously replicating sequence, phage or nucleotide sequence, linear or circular, of a single- or dou ble-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along WO 00/08172 PCT/EP99/05543 -14 with an appropriate 3' untranslated sequence into a plant cell. As used herein, "plant" refers to a photosynthetic organism, such as whole plants including algae, mosses, ferns and plant-derived tissues. "Plant de rived tissues" refers to differentiated and undif ferentiated tissues of a plant, including but not limited to roots, shoots, leaves, pollen, ovules, tubers, tassels, seeds and various forms of cells in culture such as intact cells, protoplasts, em bryos and callus tissue. Plant-derived tissues may be in planta, or in organ, tissue or cell culture. A "monocotyledonous plant" refers to a plant whose seeds have only one cotyledon, or organ of the em bryo that stores and absorbs nutrients. A "dicotyledonous plant" refers to a plant whose seeds have two cotyledons. As used herein, "transformation" refers inter alia to the processes by which cells, tissues or plants acquire properties encoded on a nucleic acid mole cule that has been transferred to the cell, tissue or plant. The terms "transformation" or "transfer" refer to methods to transfer DNA into cells includ ing, but not limited to microinjection, micropro jectile bombardment, permeabilizing the cell mem brane with various physical (e.g., electroporation) or chemical (e.g., polyethylene glycol, PEG) treat ments. The term "operably linked" refers to the chemical fusion of two or more fragments of DNA in a proper orientation, for instance sense or antisense orien tation, such that the fusion preserves or creates a WO 00/08172 PCT/EP99/05543 -15 proper reading frame, or makes possible the proper regulation of expression of the DNA sequences when transformed into plant tissue. The term "host cell" refers to a cell which has been genetically modified by transfer of a hetero logous or autologous nucleic acid sequence or its descendants still containing this sequence. These cells are also termed "transgenic cells". In the case of an autologous nucleic acid sequence being transferred, the sequence will be present in the host cell in a higher copy number than naturally occurring. The term "expression" as used herein is intended to mean the transcription and/or translation to a gene product from a gene coding for the amino acid se quence of the gene product. In the expression, a DNA chain coding for the sequence of a gene product is first transcribed to a complimentary RNA which is often an mRNA and then the thus transcribed mRNA is translated into the above mentioned gene product if the gene product is a protein. However, expres sion also includes the transcription of DNA in serted in antisense orientation to its regulatory elements. Expression, which is constitutive and further enhanced by an externally controlled pro moter fragment thereby producing multiple copies of mRNA and large quantities of the selected gene product, may also include over-production of a gene product. "Expression cassette" is used to refer to a DNA construct containing a promoter region opera bly linked to a coding sequence, which is operably linked to a 3' end and together is capable of di- WO 00/08172 PCT/EP99/05543 -16 recting a mRNA of the coding region, resulting in synthesis of a mRNA and/or a protein product in plant tissue. The term "translation start codon" or "initiation codon" refers to a unit of three nucleotides (codon) in a nucleic acid sequence that specifies the initiation of protein synthesis. The term "signal peptide" refers to the N-terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precur sor protein and which is required for its entrance into the secretory pathway. The signal peptide may be recognised by the mechanisms within the same species or unrelated plant species, necessary for direction of the peptide into the secretory path way. The signal peptide may be active in seeds, leaves, tubers and other tissues of the plant. The term "signal sequence" refers to a nucleotide se quence that encodes a signal peptide. The term "vacuole targeting signal" refers to the N-terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precur sor protein and which is required for its eventual entrance into the vacuole of a cell. The vacuole targeting signal may be recognised by the mecha nisms within the same species or unrelated plant species, necessary for direction of the peptide into the vacuole of a cell. Vacuole targeting sig nals may be active in seeds, leaves, tubers and other tissues of the plant. The term "'vacuole tar geting sequence" refers to a nucleotide sequence that encodes the vacuole targeting signal.

WO 00/08172 PCT/EP99/05543 -17 A "tissue specific promoter" refers to a sequence of DNA which provides recognition signals for RNA polymerase and/or other factors required for tran scription to start controlling expression of the coding sequence precisely within certain tissues or within certain cells of that tissue. Expression in a tissue-specific manner may be only in individual tissues or cells within tissues or in combinations of tissues. Examples may include tissue specific expression in leaves only and no other tissue within the plant, or may be in petals, ovules and stamen and no other tissues of the plant. Here, "tissue specific" is also meant to describe an ex pression in a particular tissue or cell according to which the expression takes place mainly, but not exclusively, in the tissue. Such an expression is also termed tissue abundant. "Selective expression" refers to expression almost exclusively in specific organs of the plant, in cluding leaves tubers or seeds. The term may also refer to expression at specific developmental stages in an organ, such as in early or late em bryogenesis. In addition, "selective expression" may refer to expression in specific subcellular lo cations within the cell, such as the cytosol or vacuole. The present invention is, of course, advantageous since the above nucleic acid sequences can be used to identify and obtain further nucleic acid se quences and consequently proteins from maize or other organisms, in particular plants, wherein the WO 00/08172 PCT/EP99/05543 -18 obtained nucleic acid sequences and/or proteins are involved in fatty acyl-CoA metabolism. The nucleic acid sequences of the present invention can, of course, also be used for the construction of recom binant DNA in particular expression cassettes, for transcription and/or expression in various host or ganisms. The sequences of the present invention may prove particularly useful in methods to alter the VLCFA and/or wax ester composition in host cells. Preferably, the full length clone is used for pur poses of expression of the protein. For the above mentioned purposes, of course, also the 5' and 3' non-coding DNA sequences of the present invention can be used for directing or enhancing the expres sion. The nucleic acid sequences of the present in vention may be modified using standard techniques of site specific mutation or PCR. Modifications of the sequence may also be accomplished in producing a synthetic nucleic acid sequence. Such modified sequences are also encompassed by the present in vention. For example, wobble positions in codons may be changed such that the nucleic acid sequence encodes the same amino acid sequence, or alterna tively, codons can be altered such that conserva tive amino acid substitutions result. In either case, the peptide or protein maintains the desired enzymatic activity and is thus considered part of the present invention. The present invention also relates to a vector com prising any one of the nucleic acid sequences men tioned above. Such a vector is preferably a bacte rial, yeast or a viral vector, in particular a plasmid.

WO 00/08172 PCT/EP99/05543 -19 In a preferred embodiment of the present invention, the nucleic acid sequence of the present invention, in particular the coding sequence, in sense or an tisense orientation, is operably linked to regula tory elements for directing the expression of the nucleic acid sequence, preferably in plant cells such as monocot or dicot cells or in yeast. The present invention preferably contemplates, as regu latory elements, elements that direct or enhance, in particular tissue specific, expression in the above cells. These regulatory elements may be lo cated 5', 3' or 5' and 3' of the nucleic acid se quences, in particular the coding sequence, of the present invention. Of course, for instance in that case where a genomic DNA clone according to the present invention is used, regulatory elements may also be present within the nucleic acid sequence of the present invention, in particular within an in tron. However, the regulatory element may also be an intron in its entirety. The present invention relates in a preferred em bodiment to the above mentioned vector, wherein the 5' regulatory element is a transcription initiation region, preferably a plant promoter, in particular the 35S CaMV promoter or the actin promoter of rice. However, depending upon the host and/or tar get tissue, the regulatory 5' element will vary and may include other regions from viral, plasmid or chromosomal genes. These genes may be derived from E. coli, B. subtilis, yeast or the like. Of course, other regulatory elements functional in plants, e.g. from plant genes, Agrobacterium tumefaciens and/or A.rhizogenes genes may also be used. The WO 00/08172 PCT/EP99/05543 -20 promoters may be of inducible, regulatable, or con stitutive nature. The promoter may also encompass 5' untranslated regions from foreign genes and/or translation initiation sequences. In a further embodiment, the present invention re lates to a vector, wherein the 3' regulatory ele ment is a transcription termination region, pref erably a poly A addition sequence, most preferably the poly A addition sequence of the NOS gene of Agrobacterium tumefaciens. Of course, the present invention also relates to vectors described above, which furthermore contain further regulatory elements and/or elements neces sary for the stable and/or transient integration of the nucleic acid sequence of the present invention into the genome of a host, for instance, T-DNA se quences, in particular the left, the right, or both T-DNA border sequences. In a particularly preferred embodiment of the present invention, the nucleic acid sequence of the present invention is inserted, optionally in conjunction with further regulatory elements, within the T-DNA of Agrobacterium tumefa ciens or adjacent to it. The present invention relates in a further embodi ment to a host cell transformed with any one of the above mentioned vectors, in particular to a bacte rial, yeast or plant cell, for instance a monocot or dicot host cell. In a particularly preferred em bodiment, these host cells are capable of producing a protein with the activity of a fatty acid elon gase.

WO 00/08172 PCTIEP99/05543 -21 The present invention also relates to cell cul tures, tissues or calli comprising any one of the above host cells, in particular, these cells are capable of producing a protein with the activity of a fatty acid elongase, preferably from maize. The present invention also relates to a protein with the activity of a fatty acid elongase produced by any one of the above mentioned host cells, in particular to a protein encoded by a nucleic acid sequence of the present invention. In the context of the present invention, the term "protein" refers to any sequence length of amino acid, irrespective of its length. Thus, within the present invention the term "protein" relates to peptides, polypetides and proteins. The protein of the present invention may be modified by addition of carbohydrates, fats or other proteins or peptides. The proteins of the present invention may also be modified by addition of isotopes, amino-, acyl-, allyl-, or other groups. A particularly preferred amino acid sequence of the present invention is any one of the amino acid se quences of SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37 or 39. The present invention also relates to an antibody, in particular a monoclonal or polyclonal antibody, which is reactive with the protein of the present invention. These antibodies may be used to screen expression libraries to identify clones which pro duce similar or related plant cytoplasmic proteins involved in fatty acyl-CoA metabolism.

WO 00/08172 PCT/EP99/05543 -22 The present invention also relates to a method of genetically modifying a cell, preferably a plant, bacterial or yeast cell, by transforming the cell with a vector of the present invention, whereby the nucleic acid sequence contained in the vector is expressible in the cell. Thus, the present inven tion also relates to a method of transformation of a cell, in particular a plant, bacterial or yeast cell. The cells to be transformed may be cells which do not possess an endogenous DNA sequence en coding a protein with the activity of a fatty acid elongase, in particular of a maize fatty acid elon gase. However, the present invention also relates to the transformation of cells which do possess an endogenous DNA sequence encoding a protein with the activity of a fatty acid elongase, in particular a maize fatty acid elongase. In this embodiment, the DNA sequence of the present invention is particu larly preferred under the control of regulatory elements not associated originally in the cells to be transformed with the DNA sequence of the present invention and/or the DNA sequence of the present invention is preferably used as an antisense con struct. Of course, the transformation process of the present invention is not limited only to cells themselves, but may also be applied to tissues, plant parts such as cotyledons or petioles or cal luses or embryos. The present invention also relates to plants and processes to obtain plants, wherein a DNA construct comprising a nucleic acid sequence of the present invention cloned in sense or antisense orientation under the control of appropriate regulatory ele- WO 00/08172 PCT/EP99/05543 -23 ments is transformed in a plant cell so as to eliminate or reduce the wild-type expression of an autologous or endogenous gene encoding a fatty acid elongase. Such an eliminating effect may be ob tained via antisense constructs or sense con structs, wherein the latter lead to cosuppression, for instance due to a high copy number. In particu lar, expression of the transgenic copy or copies, i.e. the transformed nucleic acid sequence, may not be necessary and the mere presence of the trans formed nucleic acid sequence(s) may be sufficient to cause alteration of the very long chain fatty acids and/or waxes amount or composition. The transformation may be carried out with plant species which are naturally susceptible to Agrobac terium tumefaciens or Agrobacterium rhizogenes in fection via methods of Agrobacterium mediated transformation. Of course, other transformation methods can also be used, such as direct uptake of DNA by microinjection or particle bombardment. Of course, any further methods can also be used, such as electroporation methods or the use of plant pathogenic viruses or plant transposable elements. After transformation, the preferred plant cells of the present invention are cultivated and regener ated to intact, fertile plants via conventional methods. The present invention of course also relates to plants comprising genetically modified cells ac cording to the present invention, capable of ex pressing and/or possessing the incorporated nucleic acids of the present invention, in particular WO 00/08172 PCT/EP99/05543 -24 seeds, embryos, calluses, cotyledons, petioles and plant tissue, harvest material and reproductive tissue derived from such a plant or used to produce a plant and still comprising at least one geneti cally modified cell. Thus, the present invention relates to seeds, plant parts and embryos, non-biologically transformed, which possess, stably integrated in the genome of their cells, a preferably heterologous, nucleic acid sequence of the present invention containing a promoter recognised by the polymerases of the cells of said seeds, plant parts or embryos and the nu cleic acid sequence of the present invention en coding a protein having a non-variety-specific en zymatic fatty acid elongase activity or part thereof or being an antisense construct. Thus, the invention also relates to plants or plant parts or plant tissue, calluses, embryos, cotyle dons or petioles, non-biologically transformed, which possess, stably integrated in the genome of their cells, a, preferably heterologous, nucleic acid sequence encoding a protein having a non variety-specific enzymatic fatty acid elongase ac tivity or part thereof or being an antisense con struct under the control of a promoter recognised by the polymerases of said cells. In the case where the nucleic acid sequence of the present invention is not heterologous but autolo gous to the transformed host, the regulatory ele ments associated with the transformed DNA sequence of the present invention and/or the orientation of WO 00/08172 PCT/EP99/05543 -25 the DNA sequence of the present invention in rela tion to its promoter and/or its integration loca tion in the genome and/or the copy number in the transformed cell are most preferably different from the untransformed host. The teaching of the present invention is therefore applicable to any plant, plant genus or plant spe cies, wherein the fatty acyl-CoA metabolism is to be modified, for instance maize, rice, wheat, bar ley, rape, peanut, oat, rye, pea, soybean, potato, sugar beet, sugar cane, sorghum, Brassica, tobacco, sunflower, carrot, tomato, cucumber, cotton, pop lar, dactylis, Festuca, Lolium, Arabidopsis, let tuce, ornamental plants, etc. Finally, the present invention relates to a method of modifying the content of very long chain fatty acid molecules, in particular with C>27, in a plant cell, wherein a plant cell is transformed with a nucleic acid sequence of the present invention and the plant cell, in particular a plant regenerated from the plant cell, is grown under conditions wherein the transformed nucleic acid sequence is expressed or wherein the transformed nucleic acid sequence is present and interferes with endogenous expression of an endogenous gene encoding a fatty acid elongase. The present invention, of course, also relates to the production of VLCFAs, in particular with C>27, or modifications of its amounts of such fatty acids in host cells. Thus, the present invention provides for such a method wherein an increased production WO 00/08172 PCT/EP99/05543 -26 of VLCFAs, in particular with C>27, in the host cell may be obtained by expression of nucleic acid sequences of the present invention. The present in vention of course also relates to such a method wherein antisense constructs or sense constructs used in cosuppression technology containing se quences of the present invention are used to reduce and/or alter the content of VLCFAs, in particular with C>27, in a host cell. Thus, the present inven tion advantageously provides new plants and plant seeds and in particular plant seed oils with a de sirable fatty acid composition. The present invention also relates to a method for producing VLCFA's, in particular with C>27, and/or waxes in a plant comprising producing a genetically modified plant of the present invention, harvesting the plant and extracting the VLCFA's and/or waxes from the harvested plant. The present invention also relates to the produc tion of wax esters or modifications of its amounts in host cells of the present invention. The host cells used for the production or modification of the amount of wax esters should contain fatty acyl and fatty alcohol substrates for the wax synthase activity of the present invention. Further preferred embodiments of the present inven tion are specified in the appended claims. The present invention is explained in more detail by the sequence listing which is part of the pres ent description.

WO 00/08172 PCT/EP99/05543 -27 SEQ. ID. No. 1 depicts the deduced full length amino acid sequence coded by ZmKCS1 having 494 amino acids. SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 34, 36 and 38 depict partial nucleotide and deduced amino acid sequences coded by ZmKCS1. In detail and in reference to the nu cleotide numbering of the full length nucleotide sequence of ZmKCS1 depicted in SEQ. ID. No. 32. SEQ. ID. No. 2 represents the nucleotides from and including position 138 to 161, SEQ. ID. No. 4 from position 171 to 215, SEQ. ID. No. 6 from position 225 to 296, SEQ. ID. No.8 from position 306 to 383, SEQ. ID. No. 10 from position 429 to 512, SEQ. ID. No. 12 from position 540 to 632, SEQ. ID. No. 14 from position 885 to 935, SEQ. ID. No. 16 from po sition 981 to 1013, SEQ. ID. No. 18 from position 1077 to 1109, SEQ. ID. No. 20 from position 1119 to 1154, SEQ. ID. No. 22 from position 1212 to 1256, SEQ. ID. No. 24 from position 1458 to 1490, SEQ. ID. No. 26 from 1524 to 1571, SEQ. ID. No. 28 from position 1593 to 1616, SEQ. ID. No. 30 from posi tion 1566 to 1580, SEQ. ID. No. 34 from position 633 to 665, SEQ. ID. No. 36 from position 1053 to 1070 and SEQ. ID. No. 38 from position from posi tion 1344 to 1358. SEQ. ID. Nos. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37 and 39 depict partial amino acid sequences of ZmKCS1 deduced from the above partial nucleotide sequences.

WO 00/08172 PCTIEP99/05543 -28 SEQ. ID. No. 32 depicts the full length nucleotide sequence from position 1 to position 1954 of clone ZmKCS1 comprising a nucleotide sequence encoding the amino acid sequence of SEQ. ID. No. 1. SEQ. ID. No. 33 exclusively depicts the coding re gion of the nucleotide sequence of SEQ. ID. No. 32. The present invention will now be more specifically described in the following examples and the accom panying drawings. Figure 1 presents a restriction map of the DNA con struct pActl.D16.3x. Figure 2 presents a restriction map of the DNA con struct pBHT9.1. Example 1: Isolation of the ZmKCS1 cDNA clone: A cDNA bank was constructed from RNA isolated from maize kernels of the line cl-p (Chen and Coe, 1977). Messenger RNA was isolated from the kernels that were germinated under light for 0, 3 and 6 hours. cDNA from this mRNA was generated with the Stratagene ZAP-cDNA-Synthesis kit following manu facturer's protocols. Upon size fractionation, the cDNA fragments longer than 500 bp were ligated into the vector kUni-ZAP XR (Stratagene) as suggested by the manufacturer. The resulting DNA molecules were packaged into phage particles using the Gigapack II-Gold Packaging Extracts as suggested by the WO 00/08172 PCT/EP99/05543 -29 manufacturer. The titer of the unamplified bank was 1,000,000 recombinant plaque forming units (pfu). This bank was amplified before screening. The differential screening of a total of 60,000 pfu of the cDNA mentioned above was performed as fol lows: twenty four plates (150 mm in diameter) were prepared with 2,500 pfu per plate. These plates were lifted twice with nitrocellulose filters. These filter-replicates were hybridised under stringent conditions, i.e. hybridisation buffer: 1 M NaCl, 1% SDS, 10% dextran sulphate, 100 pg/ml single stranded salmon sperm DNA; hybridisation temperature 650 C, first wash 2 x SSC; 0.5% SDS at room temperature; second wash: 0.2 x SSC; 0.5% SDS at 650 C, with a radioactively labelled single stranded cDNA preparation from mRNA isolated from maize kernels germinated either in the dark or in the presence of light for 3 and 6 hours, respec tively. One clone was isolated from this differential screening, D16.3, that carried an insert with high sequence identity to known KCS (James et al., The Plant Cell, March 1995 7, 309-319 and Lassner et al., The Plant Cell, February 1996 8, 281-292) genes at the nucleotide and deduced amino acid lev els. Since this clone did not contain the putative initiation codon of the longest open reading frame, 1,200,000 pfu of the same cDNA bank mentioned above were hybridised under the above described stringent conditions with the insert of the clone D16.3 in order to isolate a clone containing the complete coding region. One isolated clone, 9.1, carried an WO 00/08172 PCTIEP99/05543 -30 insert whose nucleotide sequence encompassed, with complete identity, the complete sequence of the in sert of D16.3 as well as the full coding region. This clone was renamed ZmKCS1. The full length nu cleotide sequence of clone ZmKCS1 is depicted in SEQ. ID. No. 32. The sequence contains a 5' non coding region from positions 1 to 134. A transla tion initiation codon ATG is located at position 135 to 137. The translation stop codon TGA is lo cated at position 1617 to 1619 whilst from position 1620 to position 1954 a 3' untranslated non-coding region is located. SEQ. ID. No. 33 represents those nucleotides, i.e. nucleotides 135 to 1616, which encode the amino acid sequence depicted in SEQ. ID. No. 1. SEQ. ID. Nos. 2 to 31 and 34 to 39 represent partial DNA and amino acid sequences being derived by sequence comparisons with genes which are thought to encode proteins with similar activity from Arabidopsis, jojoba, Brassica napus and Bras sica juncea. The partial nucleotide and amino acid sequences SEQ. ID. Nos. 2 to 31 and 34 to 39 of the present invention share hardly any, or no, sequence identity with corresponding known sequences from other species and are therefore particularly suit able for cloning purposes in monocotyledonous plants, in particular in maize. The cDNA clone ZmKCS1 has been used in northern ex periments with maize tissue and shows expression in germinating kernels, predominantly in endosperm tissue, and young seedlings as compared to adult leaves with almost no expression. In tassel tissue strong expression could also be observed.

WO 00/08172 PCTIEP99/05543 -31 Example 2: Modification of the VLCFA composition of plants: Generation of transgenic plants carrying a ZmKCS1 antisense or sense construct: 2.1 Transformation of monocot plants (e.g. maize): The VLCFA and/or wax composition of a maize plant may be altered by the antisense technology and clone D16.3. Upon inhibition of the expression of the ZmKCS1 gene, the length of the carbon chains of the above mentioned compounds will be significantly reduced. The 1.2-kb insert of the clone pD16.3 was cloned into a unique cloning site of the expression vector pActl.cas (Cambia TG 0063) (this vector contains the promoter and first intron of the acting gene of rice; McElroy et al., 1990) in antisense orienta tion in relation to the promoter. The resulting 5,2 kb construct, pActl.D6.3a (figure 1), was used to transform, via a particle bombardment protocol (Brettschneider, Becker and L6rz 1997), scutelar tissue of immature embryos of the hybrid maize line A188xH99. 1.0-1.4 mm long immature maize embryos were iso lated and cultured on nutrient medium. The embryos were bombarded 4 to 10 days after isolation. The bombardment was performed using a PDS 1000/He gun (BioRad) . The gold particles, 0.4-1.2 pm in diame ter, were accelerated onto the plant material using rupture disks of 1350 psi. For selection of the WO 00/08172 PCT/EP99/05543 -32 transformed tissue, a construct (p35S-PAT; P.Eckes), harbouring a selectable marker conferring resistance to the drug phosphinotricin (PPT), was co-transformed with pActl.Dl6.3. Plants were regenerated on culture media supple mented with 1 mg/ml PPT and were transferred to soil after reaching 10 cm in height. Southern blot analysis with genomic DNA isolated from transformed regenerated maize plants showed the presence of the transformed construct. The screening of the transformants involved the de termination, via Northern blots, of the effective ness of the antisense method. A reduction of the amount of ZmKCS1 mRNA could be observed. Further more, the VLCFA/wax composition was analysed by gas-chromatography. It could be shown that the lengths of the carbon chain of VLCFAs and waxes present in the transgenic maize plants were drasti cally reduced. Also, the shape of leaves was dras tically changed and most plants were male sterile. The transformed maize plants were effected primar ily in the development of the leaf structure. Leaf development showed a characteristic misformation of the outer areas of leaves. Some of the transformed plants were very small in growth and most plants were negatively effected in tassel and ear develop ment. Also, most plants did not develop intact an thers and pollen development was strongly reduced or absent. Pollen shedding was also effected and microscopical analysis of pollen grains revealed that many pollen grains were aborted.

WO 00/08172 PCT/EP99/05543 -33 Southern analysis of maize and tobacco plants re vealed that the plants were transformed and con tained sequences of the construct used. 2.2 Transformation of dicot plants (e.g. tobacco): Analogously to the experimental set-up of example 2.1, the VLCFA/wax composition of a tobacco plant was altered. The insert of the clone ZmKCS1 was cloned in a binary vector in sense orientation in relation to the 35S promoter of CaMV. The resulting 14.9 kb construct pBHT9.1 (figure 2) was trans formed into Agrobacterium tumefaciens and used to transform leaf-discs of tobacco. Transformation and regeneration of transformed plants was carried out following established protocols. Southern blot analysis with genomic DNA isolated from transformed and regenerated tobacco plants showed the presence of the transformed construct. The analysis of the transformants has been done as mentioned above. The tobacco plants obtained showed a severe reduction in the amount of ZmKCSI mRNA and a reduction in the lengths of carbon chains in the VLCFAs and waxes present in the tobacco plants ob tained. The results show that possibly a cosuppres sion effect can be obtained using a sense orienta tion of the nucleic acid sequence of the present invention.

WO 00/08172 PCT/EP99/05543 -34 Example 3: Isolation of new genes involved in fatty acid elon gation in maize through a direct/interaction ap proach: ZmKCS1 is a member of a gene family in maize. cDNA clones which are similar in the coding region but vary widely in the 3' untranslated region, have been isolated according to the present invention, signifying that they derived from different genes. The working hypothesis is that this large gene fam ily includes condensing enzymes participating in specific elongation steps and in different tissues, e.g. seed and leaf. The fact that ZmKCS1 is also expressed in leaves, allows direct probing of the condensing step of epicuticular wax biosynthesis. The genes for all other condensing enzymes charac terised to date are solely expressed in seeds. It is preferred to use an oligonucleotide compris ing the sequence 5'-GACGACTGCATCCAC-3' (SEQ ID. No. 30) to screen, via PCR, various cDNA banks con structed with mRNA from various tissues, especially from leaves of different ages, in order to find the active condensing enzymes in those tissues. The fact that the four enzyme activities catalysing fatty acid elongation interact physically in a tightly-bound complex may be used to isolate clones coding the other three enzymatic activities. It is preferred to use the "Two Hybrid Screening" (Stratagene) using ZmKCS1 and the other identified condensing enzymes as baits to isolate their inter- WO 00/08172 PCT/EP99/05543 -35 acting partners. The screening would be repeated until all four partners had been identified. Example 4: Functional analysis of the ZmKCS1 protein: expression of ZmKCS1 in yeast (Saccharomyces cerevisiae) To determine the enzymatic activity of the protein ZmKCS1, a yeast strain (INVSc-1; Invitrogen) was transformed with a construct expressing ZmKCS1. The influence that the presence of this protein in yeast cell has on the fatty acid composition was assessed. These experiments are carried out as follows by use of PCR technology, the start codon of ZmKCS1 has been modified to fit the yeast consensus (Cigan and Donahue, 1987) and a restriction site for cloning has been incorporated immediately 3' of the stop codon. The resulting fragment was cloned in pBS+ (Stratagene) to generate the construct pB9.1M. The correctness of its sequence was determined upon se quencing. The fragment from pB9.1M was cloned in the expression vector PYes2 (Invitrogen) and the resulting construct pY9.1M was then transformed in the yeast strain. In pY9.1M the expression of the ZmKCS1 gene is un der the control of a strong, galactose-inducible promoter. The transformed yeast cells were grown on a galactose-free medium and the expression of ZmKCS1 was induced upon addition of galactose. Af- WO 00/08172 PCTIEP99/05543 -36 ter further culturing, the cells were harvested and its lipids extracted and analysed through gas chromatography. Example 5: Over-expression and production of polyclonal anti bodies against the protein ZmKCS1: In order to isolate sufficient quantities of the protein ZmKCS1 to raise polyclonal antibodies, the nucleotide sequences coding for this protein was cloned in an expression vector of the series pRSET (Invitrogen). These vectors allow over-expression of proteins in E. coli under the control of the bacteriophage T7 promoter. The nucleotide sequence of ZmKCS1 was modified by PCR to allow the cloning, in frame, of the open reading frame coding for ZmKCS1 with the bacterial start codon present in the vector. The resulting construct was transformed into an E. coli strain that carries the gene encoding the bac teriophage T7 RNA polymerase under the control of the IPTG-inducible lac promoter. Addition of IPTG to the culture medium results in a strong and rapid induction of the expression of the ZmKCS1. The re sulting protein also carries an amino acid tag con sisting of six histidines. This allows the ready isolation of the tagged proteins by chromatography on niquel columns. The purified ZmKCS1 protein may be used to raise antibodies in, for example, rabbits.

WO 00/08172 PCT/EP99/05543 -37 These antibodies may be helpful in, for example, the identification of clones which express ZmKCS1 similar proteins and the determination of this pro tein in planta. References: Brettschneider, R., Becker. D. and L6rz, H. 1997. Efficient transformation of scutelar tissue of im mature maize embryos. Theor. Appl. Genet. 94: 737 748. Chen, S. and Coe, E.H. 1977. Control of anthocyanin synthesis by the C-locus in maize. Biochem. Genet ics 15: 333-346 Cigan, A.M. Donahue, T.F. 1987. Sequence and struc tural features associated with translational ini tiator regions in yeast - a review. Gene 59: 1-18. McElroy, D., Zhang, W., Cao, J. and Wu, R. 1990. Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2: 163-171.

Claims (31)

1. A nucleic acid molecule for use in cloning nu cleic acid molecules encoding a protein with the activity of a fatty acid elongase wherein the nu cleic acid molecule comprises a nucleic acid se quence selected from the group consisting of a) the nucleic acid sequence encoding any one of the amino acid sequences identified in SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37 and 39 or a complementary strand thereof, b) a nucleic acid sequence which hybridises to the nucleic acid sequence defined in a) and c) alleles or derivatives of the nucleic acid sequence defined in a) or b).

2. The nucleic acid molecule according to claim 1, which is selected from the group consisting of a) SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 36 and 38 or a complementary strand thereof, b) a nucleic acid sequence which hybridises to the nucleic acid sequence defined in a), WO00/08172 PCT/EP99/05543 -39 c) a nucleic acid sequence, which is degener ate as a result of the genetic code to the nucleic acid sequence defined in a) or b) and d) alleles or derivatives of the sequence de fined in a), b) or c).

3. The nucleic acid sequence according to any one of the preceding claims, which is derived from Zea mays.

4. A vector comprising the nucleic acid sequence of any one of the preceding claims.

5. The vector of claim 4, which is a bacterial, yeast or a viral vector, in particular a plasmid.

6. The vector of claim 5, wherein the nucleic acid sequence of any one of claims 1 to 3 is operably linked to regulatory elements for directing the ex pression of the nucleic acid sequence, preferably in a plant cell, in particular a monocot or dicot cell or a yeast.

7. The vector of claim 6, wherein the regulatory elements direct or enhance tissue specific expres sion, preferably in plants, in particular monocots.

8. The vector of any one of claims 4 to 7, wherein the regulatory elements are 5', 3' or 5' and 3' regulatory elements. WO00/08172 PCTIEP99/05543 -40

9. The vector of claim 8, wherein the 5' regulatory element is a plant promoter, in particular the 35S CaMV promoter.

10. The vector of claim 8 or 9, wherein the 3' regulatory element is a transcription termination region, preferably a poly A addition sequence, most preferably the poly A addition sequence of the NOS gene of Agrobacterium tumefaciens.

11. The vector of any one of claims 4 to 10, wherein the nucleic acid sequence is operably linked to a targeting sequence.

12. The vector according to any one of claims 4 to 11, which furthermore contains T-DNA, in particular the left, the right or both T-DNA borders.

13. The vector according to any one of claims 4 to 12, wherein the nucleic acid sequence is inserted, optionally in conjunction with the regulatory ele ments, within the T-DNA or adjacent to it.

14. A host cell transformed with the vector of any one of claims 4 to 13.

15. The host cell of claim 14, which is a plant, yeast or bacterial cell, in particular a monocot or dicot cell.

16. A cell culture, preferably a plant, yeast or bacterial cell culture, comprising a cell according to any one of claims 14 or 15. WO 00/08172 PCT/EP99/05543 -41

17. A cell culture comprising cells, preferably plant, yeast or bacterial cells, which are capable of producing a protein with the activity of a fatty acid elongase, preferably from Zea mays.

18. A protein with the activity of a fatty acid elongase produced by any one of the cells of claims 14 or 15, preferably from Zea mays.

19. A protein having the activity of a fatty acid elongase and being encoded by any one of the se quences given in claims 1 to 3.

20. An antibody, which is reactive with the protein of claim 18 or 19.

21. A plant comprising a genetically modified cell according to any one of claims 14 or 15, preferably Zea mays.

22. Seeds and plant tissue, preferably comprising a genetically modified cell according to any one of claims 14 or 15, from a plant according to claim 22.

23. A method of genetically modifying a cell by transforming the cell with a vector according to any one of claims 4 to 13, whereby the nucleic acid sequence contained in the vector is expressible in the cell.

24. The method of claim 23, wherein the cell is a plant, bacterial or yeast cell. WO00/08172 PCT/EP99/05543 -42

25. The method of claim 24, wherein the transformed cell is regenerated to a differentiated plant.

26. The method of claim 24, wherein the cell is transformed by transfer of nucleic acid sequences from a bacterium to the cell.

27. The method of claim 24, wherein the cell is transformed by direct uptake of nucleic acid se quences, by microinjection of nucleic acid se quences or by particle bombardment.

28. A method of modifying the content of very long chain fatty acid molecules and/or waxes in a plant cell, wherein a plant according to claim 21 or a plant being transformed according to any one of claims 23 to 27 is grown under conditions, wherein the transformed nucleic acid sequence is present or expressed.

29. The method of claim 28, wherein the proportion of very long chain fatty acid molecules and/or waxes is changed, in particular increased and wherein the nucleic acid sequence expresses a pro tein such as to produce from long chain fatty acid acyl-CoA molecules very long chain fatty acid mole cules and/or waxes.

30. The method of claim 28, wherein the proportion of very long chain fatty acid molecules and/or waxes from a given proportion is decreased and wherein the nucleic acid sequence is cloned in an tisense or sense in the vector and represses the WO00/08172 PCT/EP99/05543 -43 production of a protein involved in producing very long chain fatty acid molecules and/or waxes.

31. A method of producing very long chain fatty acid molecules and/or waxes in a plant, comprising growing the plant of claim 21, harvesting the plant and extracting the very long chain fatty acid mole cules and/or waxes from the harvested plant.