DE19962409A1 - Nucleic acid encoding delta6-acetylenase or desaturase, useful for producing plant oils with increased content of unsaturated fatty acids - Google Patents

Abstract

Isolated nucleic acid (I) encoding polypeptides (II) with DELTA 6-acetylenase and/or DELTA 6-desaturase activity, is new. Isolated nucleic acid (I) encoding polypeptides (II) with DELTA 6-acetylenase and/or DELTA 6-desaturase activity, is new. (I) is a 2040 bp (N1), 1467 bp (N2) or 2160 bp (N3) sequence (all given in the specification), is equivalent to N1-N3 within the degeneracy of the genetic code or is a derivative of N1-N3 encoding a polypeptide that has \- 75% amino acid (aa) homology with a 483 aa sequence (given in the specification). Independent claims are also included for the following: (a) amino acid sequences encoded by (I); (b) an expression cassette (EC) containing (I) linked to one or more regulatory sequences; (c) a vector containing (I) and EC; (d) organisms containing (I), EC or the vectors of (c); (e) preparation of unsaturated fatty acids (A) or triglycerides (TG) with increased content of (A) by introducing (I) or EC into an oil-producing organism; (f) proteins (IIa) of 172 aa or 178 aa (given in the specification); (g) production of (A) or TG by using (Ia); and (h) (A) and TG produced by method (g).

Description

The present invention relates to a method of manufacture of unsaturated fatty acids and a process for their production of triglycerides with an increased content of unsaturated Fatty acids. The invention further relates to the use of DNA sequences coding for Δ6-acetylenase / Δ6-desaturases or Δ6-desaturases for the production of a transgenic organism, preferably a transgenic plant or a transgenic micro organism with an increased content of fatty acids, oils or lipids with Δ6 triple bonds and / or Δ6 double bonds.

The invention also relates to an isolated nucleic acid sequence; an expression cassette containing a nucleic acid sequence, a vector and organisms containing at least one Nucleic acid sequence or an expression cassette. In addition, be the invention applies to unsaturated fatty acids and triglycerides with an increased content of unsaturated fatty acids and their Use.

Fatty acids and triglycerides have a wide variety of uses in the food industry, animal nutrition, cosmetics and in the pharmaceutical sector. Depending on whether it is saturated or free unsaturated fatty acids or triglycerides with an increased Content of saturated or unsaturated fatty acids are they are suitable for a wide variety of applications for example polyunsaturated fatty acids for baby food Increase in nutritional value added. Mainly the ver different fatty acids and triglycerides from microorganisms like Mortierella or from oil-producing plants such as soy, rapeseed, Sunflower and others are obtained, usually in shape of their triacylglycerides. But you can also from animals how fish are obtained. The free fatty acids are made before partly produced by saponification.

Depending on the application, oils are saturated or unsaturated th fatty acids are preferred, so are z. B. in human nutrition Lipids with unsaturated fatty acids especially polyunsaturated th fatty acids are preferred because they have a positive influence on the Cholesterol levels in the blood and therefore the possibility of one Have heart disease. They can be found in various diets Food or drug application.

Due to its positive properties, it has in the Ver There is no lack of approaches, genes involved in synthesis of fatty acids or triglycerides are involved, for which Her position of oils in different organisms with modified Making unsaturated fatty acid content available. So becomes a Δ-9 desaturase in WO 91/13972 and its US equivalent described. In WO 93/11245 a Δ-15 desaturase in WO 94/11516 claims a Δ-12 desaturase. Δ-6 desatura Sen are in WO 93/06712, US 5,614,393, WO 96/21022 and WO 99/27111. Further desaturases are for example as in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. In WO 96/13591 describes a Δ-6-palmitoyl-ACP desaturase and claimed. The biochemical characterization of the ver different desaturases is so far only inadequate occurs because the enzymes act as membrane-bound proteins only very much are difficult to isolate and characterize (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792).

A Δ-12 acetylenase is described in WO 97/37033. This enzyme can be used to _{produce unsaturated C 18} fatty acids with a triple bond. In addition to being used in food, such fatty acids can also be used for the production of polymers due to their reactivity. Sperling et al. reported at a conference (South Lake Tahoe, Canada, June 9-13, 1999) on the cloning of an enzyme that also introduces triple bonds into fatty acids. The substrates of this enzyme differ from those of Δ-12-acetylenase and the triple bond is introduced into the fatty acids at a different position by the enzyme.

In yeasts there was a shift in the fatty acid spectrum towards unsaturated fatty acids as well as an increase in Productivity can be demonstrated (see Huang et al., Lipids 34, 1999: 649-659, Napier et al., Biochem. J., Vol. 330, 1998: 611-614). The expression of the various desaturases in trans Genetic plants, however, did not show the desired success. A shift in the fatty acid spectrum towards unsaturated fat acids could be shown, but at the same time it was shown that the synthesis performance of the transgenic plants decreased significantly, that is, compared to the original plants, only lesser ones could Quantities of oils to be isolated.

There is still a great need for new genes which code for enzymes involved in the biosynthesis of unsaturated Fatty acids are involved and make it possible to combine these in one to produce technical scale.

The object was therefore to provide further enzymes for the synthesis of unsaturated conjugated fatty acids. This object was achieved by an isolated nucleic acid sequence which codes for a polypeptide with Δ-6-acetylenase and / or Δ-6-desaturase activity, selected from the group:

• a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 sequence shown,
• b) Nucleic acid sequences that arise as a result of the degenerate th genetic code from that in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 from the nucleic acid sequence shown conduct,
• c) Derivatives of those in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 nucleic acid sequence shown, the for polypeptides with of the amino acid sequences shown in SEQ ID NO: 2 code and at least 75% homology at the amino acid level without measuring the enzymatic effect of the poly peptide is significantly reduced.

Derivatives include, for example, functional homologs of encoded by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 Enzymes or their enzymatic activity, that is, enzymes that the same enzymatic reactions as those of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 catalyze enzymes encoded, to understand. These genes also allow an advantageous one Production of unsaturated fatty acids with triple bonds and / or double bonds in the Δ6 position. Among unsaturated In the following, fatty acids are monounsaturated or polyunsaturated Fatty acids that have triple bonds and / or double bonds have to understand. The triple and / or double bonds can be conjugated or unconjugated. the mentioned in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 Sequences code for a new enzyme that produces an acetylenase and / or have Δ6-desaturase activity.

The enzyme according to the ^{invention Δ 6 -acetylenase / Δ 6} -desaturase leads before geous in fatty acid residues of glycerolipids a cis double bond in position C ₆ -C ₇ and / or converts an existing cis double bond in position C ₆ -C ₇ into a triple bond (see SEQ ID NO: 1 or SEQ ID NO: 3). The enzyme also has a Δ6-desaturase activity, which advantageously only introduces a cis double bond in position C ₆ -C ₇ in fatty acid residues of glycerolipids. The enzyme with the sequence mentioned in SEQ ID NO: 11 also has this activity. Which is a monofunctional Δ6-desaturase.

The nucleic acid sequence (s) according to the invention (for the Registration should include the singular and the plural and vice versa) or fragments thereof can be advantageous for the isolation of further genomic sequences can be used via homology screening.

The derivatives mentioned can be derived from others, for example Organisms like eukaryotic organisms like plants specifically Isolate mosses, dinoflagellates or mushrooms.

Furthermore, under derivatives or functional derivatives are the mentioned in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 To understand sequences, for example, allele variants that min at least 70% homology at the deduced amino acid level partly at least 75% homology, preferably at least 80% Homology, particularly preferably at least 85% homology, whole particularly preferably 90% homology. The homology was calculated over the entire amino acid range. It became that Program PileUp, BESTFIT, GAP, TRANSLATE or BACKTRANSLATE (= Part of the program package UWGCG, Wisconsin Package, Version 10.0-UNIX, January 1999, Genetics Computer Group, Inc., Deverux et al., Nucleic. Acid Res., 12, 1984: 387-395) was used (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153). The one derived from the nucleic acids mentioned Amino acid sequences are sequence SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 12 can be found. Identity is to be understood under homology stand, i.e. the amino acid sequences are at least 70% identical. The sequences of the invention are based on nucleic acid flat at least 65% homolog, preferably at least 70%, be particularly preferably 75%, very particularly preferably at least 80%.

Allele variants include in particular functional variants that by deletion, insertion or substitution of nucleotides as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 th sequence are available, the enzymatic activity of the derived synthesized proteins is preserved.

Such DNA sequences can be derived from the in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 DNA sequences or parts of these sequences, for example with conventional hybridization methods or the PCR technique from other eukaryotes such as those mentioned above isolate. These DNA sequences hybridize under standard conditions with the sequences mentioned. Becoming hybridized advantageously short oligonucleotides, for example the conservative fourth areas that can be compared with other acetylenase and / or desaturase genes in a manner known to the person skilled in the art can be determined. The Histidine box sequences used. But it can also be longer Fragments of the nucleic acids according to the invention or the full permanent sequences can be used for hybridization. Depending on the nucleic acid used: oligonucleotide, longer Fragment or complete sequence or whichever one Nucleic acid type DNA or RNA used for hybridization these standard conditions vary. So are enclosed For example, the melting temperatures for DNA: DNA hybrids approx. 10 ° C lower than that of DNA: RNA hybrids of the same length.

Under standard conditions, for example, depending on the nucleic acid re temperatures between 42 and 58 ° C in an aqueous buffer solution with a concentration between 0.1 to 5 × SSC (1 × SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in Presence of 50% formamide such as 42 ° C in 5 × SSC, To understand 50% formamide. The Hybridization conditions for DNA: DNA hybrids at 0.1 × SSC and Temperatures between about 20 ° C to 45 ° C, preferably between about 30 ° C to 45 ° C. For DNA: RNA hybrids, the hybridization lies conditions advantageous at 0.1 × SSC and temperatures between about 30 ° C to 55 ° C, preferably between about 45 ° C to 55 ° C. These The temperatures given for the hybridization are exemplary calculated melting temperature values for a nucleic acid he length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for the DNA hybridization are described in relevant textbooks of the Genetics such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be found according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + Calculate C content. More information on hybridization can be found those skilled in the art can find the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford. Uni versity Press, Oxford; Brown (ed), 1991, Essential Molecular Bio logy: A Practical Approach, IRL Press at Oxford University Press, Oxford.

Furthermore, homologues of the sequence SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11, for example eukaryotic Homologous, truncated sequences, single-stranded DNA of the coding and non-coding DNA sequence or RNA of the coding and Understand non-coding DNA sequence.

In addition, homologues of the sequences SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 derivatives such as, for example Understand promoter variants. These variants can be carried out by a or several nucleotide exchanges, by insertion (s) and / or Deletion (s) may be changed, but without affecting the functionality or effectiveness of the promoters are impaired. Further can the promoters by changing their sequence in their Effectiveness increased or completely through more effective promoters alien organisms are exchanged.

Derivatives are also advantageously understood to mean variants, their nucleotide sequence in the range -1 to -2000 before the start codon were changed so that the gene expression and / or the protein expression is changed, preferably increased. Furthermore are under Derivatives also understand variants that changed at the 3 'end became.

Derivatives are also to be understood as meaning the antisense DNAs which for inhibiting the protein biosynthesis of the proteins according to the invention can be used. These antisense DNAs are among the non-functional derivatives according to the invention, such as derivatives that have no enzymatic activity. More to the expert be known methods of producing non-functional derivatives are the so-called cosuppression, the use of ribozymes and introns. Ribozymes are catalytic RNA molecules with ribo nuclease activity, the single-stranded nucleic acids such as mRNA, too which they have a complementarity, intersect. With the help of these ribozymes (Haselhoff and Gerlach, Nature, 334, 1988: 585-591) mRNA transcripts catalytically sat are cleaved and thus the translation of this mRNA is suppressed will. Such ribozymes can be specifically designed for their tasks (US 4,987,071; US 5,116,742 and Bartel et al., Science 261, 1993: 1411-1418). With the help of antisense DNA can thereby increase fatty acids, lipids or oils Proportion of saturated fatty acids.

The nucleic acid sequences according to the invention, which for a Encode Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase, can be synthetically produced or naturally obtained or a mixture of synthetic and natural DNA components contained, as well as from various heterologous Δ6-acetylenase / Δ6- Desaturase and / or Δ6-desaturase gene segments of different Organisms exist. In general, synthetic nucleo tid sequences with codons derived from the corresponding Host organisms such as plants are preferred. this usually leads to an optimal expression of the heterologous Genes. These codons preferred by plants can be selected from codons with the highest protein abundance found in the most interesting plant species are expressed. An at The game for Corynebacterium glutamicum is given in: Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118. The implementation of such Experiments can be carried out using standard methods and are known to those skilled in the art.

Functionally equivalent sequences for the Δ6-acetylenase / Δ6- Coding desaturase and / or Δ6-desaturase gene are such Derivatives of the sequences according to the invention, which differ despite the nucleotide sequence still has the desired functions, that is possess the enzymatic activity of the proteins. Functional Equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. B. by chemical synthesis obtained from the codon usage of a plant adapted, artificial nucleotide sequences.

In addition, artificial DNA sequences are suitable as long as they as described above, the desired property for example increasing the content of Δ6 triple bonds or Δ6 double bonds in fatty acids, oils or lipids in the Plant by overexpression of the Δ6-acetylenase / Δ6-desaturase and / or mediate Δ6-deasaturase gene in crop plants. Such Artificial DNA sequences can, for example, by back over set of proteins constructed by means of molecular modeling, which Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase activity have or can be determined by in vitro selection. Possibly liche techniques for in vitro evolution of DNA for change or improvement of the DNA sequences are described in Patten, P.A. et al., Current Opinion in Biotechnology 8, 724-733 (1997) or in Moore, J. C. et al., Journal of Molecular Biology 272, 336-347 (1997). Coding DNA sequences are particularly suitable, by back translation of a polypeptide sequence according to the for the host plant specific codon usage can be obtained. The specific codon usage can be related to plant genetic Methods familiar to specialists through computer evaluations of others, easily identify known genes of the plant to be transformed.

Further suitable equivalent nucleic acid sequences are to be mentioned sequences which code for fusion proteins, where Part of the fusion protein is a Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase polypeptide or a functionally equivalent valent part of it. The second part of the fusion protein can e.g. B. Another polypeptide with enzymatic activity or an antigenic polypeptide sequence with their help a detection of Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase expression is possible (e.g. myc-tag or his-tag). However, this is preferably a regulatory one Protein sequence, e.g. B. a signal sequence for the ER that the Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase protein directs the desired site of action.

The Δ6-acetylenase / Δ6-desaturase or Δ6-desaturase genes in the method according to the invention with further Fatty acid biosynthesis genes are combined. examples for such genes are the acetyltransferases, further desaturases or elongases. For in-vivo and especially in-vitro synthesis is the combination with z. B. NADH cytochrome B5 reductases advantageous to take up or release the reduction equivalents can.

Among the amino acid sequences according to the invention are proteins understand the one in the sequences SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 12 or an amino acid sequence shown therefrom by substitution, inversion, insertion or deletion sequence obtainable from one or more amino acid residues ent hold, the enzymatic activity of the in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 12 obtained remains or is not significantly reduced. Under not essential Lich reduced are all enzymes to be understood that are still at least 10%, preferably 20%, particularly preferably 30% of the enzymatic Exhibit activity of the parent enzyme. Here, for example wise certain amino acids by those with similar physiko chemical properties (space filling, basicity, hydrophobizi ity etc.) can be replaced. For example, arginine residues are ge gene lysine residues, valine residues against isoleucine residues or asparagine acid residues exchanged for glutamic acid residues. But it can also one or more amino acids in their order ver exchanged, added or removed, or several these measures can be combined with one another.

Derivatives are also to be understood as functional equivalents, which in particular also include natural or artificial mutations of a originally isolated for a Δ6-acetylenase / Δ6-desaturase- and / or Δ6-desaturase coding sequence which continue to have the desired function, that is, its enzymatic function Activity is not significantly reduced. Mutations include substitutions, additions, deletions, interchanges or insertions of one or more nucleotide residues. Thus become for example, such nucleotide sequences by the before Lying invention includes, which can be obtained by modifying the D6 acetylenase / Δ6 desaturase and / or Δ6 desaturase nucleotide sequence receives. The aim of such a modification can be, for. B. the further delimitation of the coding sequence contained therein or z. B. also the insertion of further restriction enzyme cuts be put.

Functional equivalents are also variants whose Function compared to the parent gene or gene fragment, is weakened (= not significantly reduced) or increased (= Enzyme activity stronger than the activity of the starting enzyme, that is, activity is higher than 100%, preferably higher than 110%, particularly preferably higher than 130%).

The nucleic acid sequence can advantageously for example be a DNA or cDNA sequence. For insertion in an er coding Se suitable for the expression cassette according to the invention sequences are, for example, those for a Δ6-acetylenase / Δ6- Desaturase and / or Δ6-desaturase with those described above Sequences encode and which the host the ability to over production of fatty acids, oils or lipids with triple Give bonds and / or double bonds in the Δ6 position. These sequences can be of homologous or heterologous origin.

Under the expression cassette according to the invention (= nucleic acid construct or fragment) are those in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 sequences mentioned, which appear as a result of the genetic code and / or its functional or not functional derivatives to understand those with one or more Regulation signals advantageously to increase the gene expression have been functionally linked and which the Expression control the coding sequence in the host cell. This regu latory sequences are intended to target the expression of genes and enable protein expression. This can vary, for example after host organism mean that the gene only after induction is expressed and / or overexpressed, or that it is immediately ex is primed and / or overexpressed. For example, it acts in these regulatory sequences to sequences to the Inductors or repressors bind and so the expression of the Regulate nucleic acid. In addition to these new regulations sequences or instead of these sequences, the natural Regulation of these sequences before the actual structural genes still exist and possibly genetically modified be so that the natural regulation switched off and the Expression of the genes was increased. But the gene construct can also be set up more simply, i.e. no additional ones were required Regulation signals in front of the nucleic acid sequence or its Deri vate inserts and the natural promoter with its regulation was not removed. Instead, the natural regu lation sequence mutated in such a way that regulation no longer takes place and / or gene expression is increased. This changed promo gates can be in the form of partial sequences (= promoter with parts of the nucleic acid sequences according to the invention) also exist alone the natural gene can be brought to increase activity. The gene construct can also advantageously include an or several so-called "enhancer sequences" functionally linked containing the promoter, which increases expression of the Enable nucleic acid sequence. Also at the 3 'end of the DNA Sequences can insert additional advantageous sequences become like other regulatory elements or terminators. The Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase genes can be in one or more copies in the expression cassette (= Gene construct) should be included.

The regulatory sequences or factors can be like described above preferably the gene expression of the introduced Influence genes positively and thereby increase them. So a Ver strengthening the regulatory elements advantageously on the Transcription levels take place by using strong transcription signals how promoters and / or "enhancers" are used. Next to it is but also a strengthening of the translation is possible by at for example, the stability of the mRNA is improved.

Suitable promoters in the expression cassette are in principle all promoters which can advantageously control the expression of foreign genes in organisms in plants or fungi. A plant promoter or promoters derived from a plant virus are preferably used in particular. Before advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI ^q , T7, T5, T3, gal, trc, ara, SP6, λ-P _R - or contained in the λ-P _L promoter, which are advantageously used in gram-negative bacteria. Further advantageous regulatory sequences are, for example, in the gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters such as CaMV / 35S [ Franck et al., Cell 21 (1980) 285-294], SSU, OCS, lib4, STLS1, B33, nos (= nopaline synthase promoter) or contained in the ubiquitin promoter. The expression cassette can also contain a chemically inducible promoter by means of which the expression of the exogenous Δ6-ACETYLENASE / Δ6-DESATURASE and / or Δ6-DESATURASE gene in the organisms can advantageously be controlled in the plants at a certain point in time. Such advantageous plant promoters are, for example, the PRP1 promoter [Ward et al., Plant. Mol. Biol. 22 (1993), 361-366], one inducible by benzenesulfonamide (EP 388186), one inducible by tetracycline (Gatz et al., (1992) Plant J. 2, 397-404), a by Salicylic acid inducible promoter (WO 95/19443), a promoter inducible by abscisic acid (EP 335528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334). Further plant promoters are, for example, the promoter of the cytosolic FBPase from potato, the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445-245), the promoter of the phosphoribosyl pyrophosphate amidotransferase from Glycine max (see also Genbank Accession number U87999) or a node-specific promoter as in EP 249676 can advantageously be used. Particularly advantageous are those vegetable promoters that ensure expression in tissues or plant parts / organs in which fatty acid biosynthesis or its precursors take place, for example in the endosperm or in the developing embryo. Particular mention should be made of advantageous promoters which ensure seed-specific expression, such as, for example, the USP promoter or derivatives thereof, the LEB4 promoter, the phaseolin promoter or the napin promoter. The particularly advantageous USP promoter or its derivatives mediate gene expression very early in seed development (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67). Further advantageous seed-specific promoters which can be used for monocotyledonous and dicotyledonous plants are the promoters suitable for dicotyledons, such as the napingen promoter from rapeseed (US Pat. No. 5,608,152), the oleosin promoter from Arabidopsis (WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or the legume B4 promoter (LeB4, Baeumlein et al., Plant J., 2, 2, 1992: 233-239) or for Monocot-suitable promoters such as the promoters of the lpt2 or lpt1 gene from barley (WO 95/15389 and WO 95/23230) or the promoters of the barley hordein gene, the rice glutelin gene, the rice oryzin gene, des Rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene or the rye secalin gene, which are described in WO 99/16890.

Furthermore, those promoters are particularly preferred which ensure expression in tissues or parts of plants, in which for example the biosynthesis of fatty acids, oils and Lipids or their precursors takes place. To be mentioned in particular are promoters that ensure seed-specific expression Afford. Mention should be made of the promoter of the napin gene from oilseed rape (US 5,608,152), the USP promoter from Vicia faba (USP = unknown Seed protein, Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), of the oleosin gene from Arabidopsis (WO 98/45461), des Phaseolin promoter (US 5,504,200) or the promoter of legumin B4 gene (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9). Also to be mentioned are promoters such as that of the lpt2 or barley lpt1 gene (WO 95/15389 and WO 95/23230) described in monocotyledonous plants mediate seed-specific expression.

In the expression cassette (= gene construct, nucleic acid construct), as described above, other genes that are in the organisms are to be introduced. These Genes can be under separate regulation or under the same Regulatory region like the genes of the Δ6-ACETYLENASE / Δ6-DESATURASE- and / or Δ6-DESATURASE. These genes are what it is all about for example, further biosynthetic genes are advantageous fatty acid biosynthesis, which enables increased synthesis lichen. For example, let the genes for the Δ15-, Δ12-, Δ9-, Δ6-, Δ5-desaturase, the various hydroxylases that Δ12-acetylenase, the acyl-ACP-thioesterases, β-ketoacyl-ACP- Called synthases or β-ketoacyl-ACP reductases. Advantageous the desaturase genes are used in the nucleic acid construct.

In principle, all natural promoters can use their Regulatory sequences such as those mentioned above for the fiction according to the expression cassette and the method according to the invention, as described below. In addition, you can synthetic promoters can also be used advantageously.

When preparing an expression cassette, various DNA fragments can be manipulated into a nucleotide sequence which reads in the correct direction for convenience and which has a correct reading frame. For the Connection of the DNA fragments (= nucleic acids according to the invention) adapters or linkers can be attached to the fragments are set.

Appropriately, the promoter and terminator Regions in the direction of transcription with a linker or Polylinker that contains one or more restriction sites for the Insertion of this sequence contains, are provided. Usually the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6 restriction sites. In general, the linker has within the regulatory areas are less than 100 bp in size, often less than 60 bp, but at least 5 bp. The promoter can be native or homologous as well as strange or heterologous be to the host organism, for example, to be the host plant. The ex compression cassette contains in the 5'-3 'direction of transcription the promoter, a DNA sequence that for a Δ6-acetylenase / Δ6- Desaturase and / or Δ6-desaturase gene encoded and a region for transcriptional termination. Different termination areas can be interchanged as required.

Furthermore, tampering, the matching restriction cut provide or the superfluous DNA or Restrik Remove tion interfaces are used. Where insertions, Deletions or substitutions such as B. Transitions and Trans versions are possible, in vitro mutagenesis, primer repair, restriction or ligation can be used. With geeig Neten manipulations, such as. B. Restriction, -chewing-back- or Filling in overhangs for -bluntends- can be complementary Ends of the fragments made available for ligation will.

Of importance for an advantageously high expression can inter alia. the attachment of the specific ER retention signal SEKDEL (Schouten, A. et al., 1996 Plant Mol. Biol. 30: 781-792), the average expression level is thus tripled up to quadrupled. There can also be other retention signals that occur naturally licher, in the case of plant and animal ones located in the ER Proteins are used to build the cassette will.

Preferred polyadenylation signals are vegetable poly adenylation signals, preferably those that are essentially T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular of gene 3 of the T-DNA (octopine synthase) des Ti plasmids correspond to pTiACH5 (Gielen et al., EMBO J.3 (1984), 835 ff) or corresponding functional equivalents.

An expression cassette is produced by fusion a suitable promoter with a suitable Δ6-acetylenase / Δ6- Desaturase and / or Δ6 desaturase DNA sequence and one Polyadenylation signal after common recombination and Cloning techniques, such as those in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

When preparing an expression cassette, various DNA fragments can be manipulated into a nucleotide sequence which reads in the correct direction for convenience and which has a correct reading frame. For the Connection of the DNA fragments to one another can be attached to the fragments Adapters or linkers are attached.

Appropriately, the promoter and terminator Regions in the direction of transcription with a linker or Polylinker that contains one or more restriction sites for the Insertion of this sequence contains, are provided. Usually the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6 restriction sites. In general, the linker has within the regulatory areas are less than 100 bp in size, often less than 60 bp, but at least 5 bp. The promoter can be native or homologous as well as strange or heterologous to be the host plant. The expression cassette contains in the 5'-3 'direction of transcription the promoter, a DNA sequence the encodes a D6 acetylenase / desaturase gene and a region for transcriptional termination. Different termination areas can be interchanged as required.

When preparing an expression cassette, various DNA fragments can be manipulated into a nucleotide sequence which reads in the correct direction for convenience and which has a correct reading frame. For the Connection of the DNA fragments to one another can be attached to the fragments Adapters or linkers are attached.

Appropriately, the promoter and terminator Regions in the direction of transcription with a linker or Polylinker that contains one or more restriction sites for the Insertion of this sequence contains, are provided. Usually the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6 restriction sites. In general, the linker has within the regulatory areas are less than 100 bp in size, often less than 60 bp, but at least 5 bp. The promoter can be native or homologous as well as strange or heterologous to be the host plant. The expression cassette contains in the 5'-3 'direction of transcription the promoter, a DNA sequence the for a Δ6-acetylenase / Δ6-desaturase or Δ6-desaturase gene encoded and a region for transcriptional termination. Different termination areas are arbitrary against each other interchangeable.

The DNA sequence coding for a Δ6-acetylenase / Δ6-desaturase- and / or Δ6-desaturase from Ceratodon purpureus includes all Sequence features that are necessary to locate the fat acid, lipid or oil biosynthesis to correct localization reach. Therefore, there are no other targeting sequences per se necessary. However, such localization can be desirable and be advantageous and therefore artificially altered or amplified so that such fusion constructs are also preferred Partial embodiment of the invention are.

Particularly preferred are sequences that targeting in Ensure plastids. In certain circumstances it can also targeting in other compartments (referenced: Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423) e.g. B. in the vacuole, into the mitochondrion, into the endoplasmic reticulum (ER), Peroxisomes, lipid bodies or due to the lack of corresponding operative sequences remain in the compartment of origin, the cytosol, be desirable.

The nucleic acids according to the invention are advantageously sequences together with at least one reporter gene into one Expression cassette cloned into the organism via a Vector or directly into the genome. This Reporter gene should be easily detectable via a growth, fluorescence, chemo, bioluminescence or resistance assay or via a photometric measurement. At Antibiotic or herbicides are playful as reporter genes resistance genes, hydrolase genes, fluorescent protein genes, bio luminescence genes, sugar or nucleotide metabolism genes or bio synthesis genes such as the Ura3 gene, the Ilv2 gene, the luciferase gene, the β-galactosidase gene, the gfp gene, the 2-deoxyglucose-6- phosphate phosphatase gene, the β-glucuronidase gene, β-lactamase gene, the neomycin phosphotransferase gene, the hygromycin phosphotrans ferase gene or the BASTA (= gluphosinate resistance) gene. These genes allow easy measurability and quantification availability of the transcription activity and thus the expression of the Genes. This can be used to identify genome locations that have a show different productivity.

According to a preferred embodiment, an expression comprises upstream cassette, d. H. at the 5 'end of the coding sequence, a promoter and downstream, d. H. at the 3 'end, a polyadeny lation signal and possibly other regulatory ele elements which, with the intervening coding sequence for the Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase DNA se are operatively linked. Under an operational link one understands the sequential arrangement of promoter, coding Sequence, terminator and possibly other regulatory elements of the art that each of the regulative elements has its function in the Fulfill expression of the coding sequence as intended can. The preferred sequences for operative linkage are Targeting sequences to ensure the subcellular locales sation in plastids. But also targeting sequences to guarantee performance of subcellular localization in the mitochondrion, im Endoplasmic reticulum (= ER), in the cell nucleus, in oil bodies or other compartments can be used if necessary as well Translation enhancers such as the 5 'guide sequence from the tobacco Mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).

An expression cassette can, for example, be a constitutive Promoter (preferably the USP or napin promoter), the to expressing gene and the ER retention signal. as ER retention signal is preferably the amino acid sequence KDEL (Lysine, aspartic acid, glutamic acid, leucine) are used.

For expression in a prokaryotic or eukaryotic host organism, for example a microorganism such as a fungus or a plant, the expression cassette is advantageously inserted into a vector such as a plasmid, a phage or other DNA that enables optimal expression of the genes in the host organism. Suitable plasmids are, for example, in E. coli pLG338, pACYC184, pBR series such as. B. pBR322, pUC series such as pUC18 or pUC19, M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ¹¹³ -B1, λgt11 or pBdCI, in Streptomyces pIJ7024., PIJ364., PIJ364 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL2 or pBB116, further advantageous fungus vectors are described by Romanos, MA et al., [(1992) "Foreign gene expression in yeast: a review ", Yeast 8: 423-488] and by von den Hondel, CAMJJ et al. [(1991) "Heterologous gene expression in filamentous fungi] and in More Gene Manipulations in Fungi [JW Bennet & LL Lasure, eds., P. 396-428: Academic Press: San Diego] and in" Gene transfer systems and vector development for filamentous fungi "[von den Hondel, CAMJJ & Punt, PJ (1991) in: Applied Molecular Genetics of Fungi, Peberdy, JF et al., eds., p. 1-28, Cambridge University Press: Cambridge]. Advantageous Yeast promoters are, for example, 2 µM, pAG-1, YEp6, YEp13 or pEMBLYe23. Examples of algae or plant promoters are pLGV23, pGHlac ⁺ , pBIN19, pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer, L., 1988) The abovementioned vectors or derivatives of the abovementioned vectors represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York- Oxford, 1985, ISBN 0 444 904018) ete plant vectors are, inter alia, in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, pp. 71-119. Advantageous vectors are so-called shuttle vectors or binary vectors that replicate in E. coli and Agro bacterium.

In addition to plasmids, vectors are also all other subjects known vectors such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, Phasmids, phagemids, cosmids, linear or circular DNA too to understand. These vectors can be autonomous in the host organism replicated or chromosomally replicated is preferred a chromosomal replication.

In a further embodiment of the vector, the Expression cassette according to the invention also advantageously in Form a linear DNA to be introduced into the organisms and via heterologous or homologous recombination into the genome of the Host organism are integrated. This linear DNA can be made from a linearized plasmid or just from the expression cassette as a vector or the nucleic acid sequences according to the invention exist.

In a further advantageous embodiment, the nucleic acid sequence according to the invention also alone in a Organism are introduced.

Should, in addition to the nucleic acid sequence according to the invention, further Genes are introduced into the organism so they can all go together with a reporter gene in a single vector or each single gene with a reporter gene in one vector each in the Organism are introduced, with the various vectors can be introduced simultaneously or successively.

The vector advantageously contains at least one copy of the nucleic acid sequences according to the invention and / or the invention according to the expression cassette.

For example, the plant expression cassette can be incorporated into the tobacco transformation vector pBinAR. Fig. 1 shows the tobacco transformation vectors pBinAR with 35S promoter (C) and pBin-USP with the USP promoter (D). The output vectors are shown in Fig. 1A) and B).

Alternatively, a recombinant vector (= expression vector) also be transcribed and translated in vitro, e.g. B. by Use of the T7 promoter and the T7 RNA polymerase.

Expression vectors used in prokaryotes make frequent use inducible systems with and without fusion proteins or fusion oligopeptides, these fusions being both N-terminal and C-terminal or other usable domains of a protein can. Such fusion vectors are usually used to: i.) The Increase the expression rate of the RNA ii.) The achievable protein increase synthesis rate, iii.) increase the solubility of the protein increase, iv.) or purification by one for affinity to simplify the binding sequence usable for chromatography. Frequently also become proteolytic cleavage sites via fusion proteins introduced what is the cleavage of part of the fusion protein also allows cleaning. Such recognition sequences for Recognize proteases are z. B. Factor Xa, Thrombin and Entero kinase.

Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Inc., Smith, D.B. and Johnson, K.S. (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which glutathione S-transferase contains (GST), maltose binding protein, or protein A.

Further examples of E. coli expression vectors are pTrc [Amann et al., (1988) Gene 69: 301-315] and pET vectors [Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Stratagene, Amsterdam, Netherlands].

Further advantageous vectors for use in yeast are pYepSec1 (Baldari, et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES derivatives (Invitrogen Corporation, San Diego, CA). Vectors for use in fila mentous mushrooms are described in: von den Hondel, C. A. M. J. J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., Eds., P. 1-28, Cambridge University Press: Cambridge.

Alternatively, insect cell expression can also be advantageous vectors are used z. B. for expression in Sf 9 cells. These are e.g. B. the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

Furthermore, plants can advantageously be used for gene expression cells or algae cells are used. Examples of plants Expression vectors can be found in Becker, D., et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border, "Plant Mol. Biol. 20: 1195-1197 or in Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation ", Nucl. Acid. Res. 12: 8711-8721.

Furthermore, the nucleic acid sequences according to the invention can expressed in mammalian cells. Example of corresponding Expression vectors are named pCDM8 and pMT2PC in: Seed, B. (1987) Nature 329: 840 or Kaufman et al. (1987) EMBO J. 6: 187-195). Promoters to be used are preferably viral Origin such as B. promoters of the polyoma, adenovirus 2, cyto megalovirus or Simian Virus 40. More prokaryotic and eukaryotic expression systems are mentioned in Chapter 16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

The introduction of the nucleic acids according to the invention, the Expression cassette or the vector in organisms, for example wise in plants can in principle according to any one skilled in the art known methods.

The person skilled in the art can use appropriate methods for microorganisms Textbooks by Sambrook, J. et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by F. M. Ausubel et al. (1994) Current protocols in molecular bio logy, John Wiley and Sons, by D. M. Glover et al., DNA Cloning Vol. 1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Laboratory Press or Guthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press.

The transfer of foreign genes into the genome of a plant is made referred to as transformation. The described Methods for the transformation and regeneration of plants Plant tissues or plant cells for transient or stable Transformation used. Suitable methods are the protoplasts transformation by polyethylene glycol-induced DNA uptake, the biolistic process with the gene gun - the so-called particle bombardment method, electroporation, incubation dry embryos in a solution containing DNA, microinjection and Agrobacterium-mediated gene transfer. The called th methods are, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). Preferably the construct to be expressed is cloned into a vector, which is suitable to transform Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed with such a vector can then in a known manner for the transformation of plants, in particular of crops, such as. B. of tobacco plants, used by, for example, wounded leaves or Leaf pieces bathed in an agrobacterial solution and then cultivated in suitable media. The transformation of Plants with Agrobacterium tumefaciens is for example from Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 be written or is known, inter alia, from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

Transformed with an expression vector according to the invention Agrobacteria can also be used in a known manner for trans formation of plants such as test plants such as Arabidopsis or Cultivated plants such as cereals, maize, oats, rye, barley, wheat, Soy, rice, cotton, sugar beet, canola, sunflower, flax, Hemp, potato, tobacco, tomato, carrot, paprika, rapeseed, tapioca, Cassava, arrowroot, marigolds, alfalfa, lettuce and the various Tree, nut and wine species, especially from oil-bearing cultures plants like soy, peanut, castor oil, sunflower, corn, tree wool, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa beans can be used, e.g. B. by ver sore leaves or pieces of leaf in an agrobacterial solution bathed and then cultivated in suitable media.

The genetically modified plant cells can do all that Methods known to those skilled in the art are regenerated. Appropriate Methods can be found in the above-mentioned publications by S. D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer can be taken.

As organisms or host organisms for the inventive Nucleic acid, the expression cassette or the vector are suitable in principle, all organisms that are capable are advantageous Fatty acids specifically synthesize unsaturated fatty acids or are suitable for the expression of recombinant genes. Examples are plants such as Arabidopsis, Asteraceae such as Calendula or crops such as soy, peanut, castor, suntan flower, corn, cotton, flax, rapeseed, coconut, oil palm, dyer safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as mushrooms, for example, the genus Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae or Protozoa like dinoflagellates called like Crypthecodinium. Before Attention is drawn to organisms that naturally produce oils in larger ones Can synthesize quantities like mushrooms like Mortierella alpina, Pythium insidiosum or plants such as soy, rapeseed, coconut, oil palm, safflower, castor, calendula, peanut, cocoa bean or Sunflower or yeasts like Saccharomyces cerevisiae, especially Soy, rapeseed, sunflower, calendula or are preferred Saccharomyces cerevisiae. In principle, as host organisms transgenic animals are also suitable, for example C. elegans.

Usable host cells are also mentioned in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

Usable expression strains e.g. B. those that have a lower Have protease activity are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.

Another object of the invention relates to the use an expression cassette containing DNA sequences coding for a Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase gene or with these hybridizing DNA sequences for the transformation of Plant cells, tissues or parts of plants. Purpose of use is the increase in the content of fatty acids, oils or lipids with an increased content of triple bonds and double bonds in Δ6 position.

Depending on the choice of promoter, the expression of the Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase gene specifically in leaves, seeds, tubers or other parts of the plant. Such fatty acids, or oils Lipids with Δ6 triple bonds or Δ6 double bonds over producing transgenic plants, their propagation material, as well as their plant cells, tissues or parts are another Subject of the present invention. A preferred one subject matter according to the invention are containing transgenic plants a functional or non-functional according to the invention (= Antisense DNA or enzymatic inactive enzyme) nucleus acid sequence or functional or non-functional Expression cassette.

The expression cassette or the nucleus according to the invention acid sequences containing a Δ6-acetylenase / Δ6-desaturase- and / or Δ6-desaturase gene sequence can also be used for Transformation of the organisms exemplified above such as Bacteria, cyanobacteria, yeast, filamentous fungi, ciliates and algae with the aim of increasing the content of fat acids, oils or lipids with Δ6 triple bonds or Δ6 double bonds are used.

Increase in the content of fatty acids, oils or lipids with Δ6 triple bonds or Δ6 double bonds means in the context of the present invention, for example, the artificially acquired Ability to increase biosynthesis through functional overexpression of the Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase gene in the organisms according to the invention partly in the transgenic plants according to the invention At least the non-genetically modified parent plants for the duration of at least one generation of plants.

The biosynthesis site of fatty acids, oils or lipids, for example wise is generally the seed or cell-layers of the seed, so that a seed-specific expression of the Δ6-acetylenase / Δ6- Desaturase and / or Δ6 desaturase gene is useful. It is However, it is obvious that the biosynthesis of fatty acids, oils or Lipids need not be limited to the seed tissue, but rather also in all other parts of the plant - for example in Epidermal cells or in the tubers - tissue-specific can.

In addition, there is a constitutive expression of the exogenous Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase gene from Advantage. On the other hand, however, an inducible expression can also be used appear desirable.

The effectiveness of the expression of the transgene Δ6-acetylenase / Δ6- Desaturase and / or Δ6-desaturase gene can, for example, in can be determined in vitro by propagation of shoot meristems. In addition, can a change in type and level of expression of the Δ6-acetylenase / Δ6- Desaturase and / or Δ6 desaturase gene and their effects on the fatty acid, oil or lipid biosynthesis performance on test plants are tested in greenhouse experiments.

The invention also relates to transgenic plants, transformed with an expression cassette containing a Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase gene sequence or DNA sequences hybridizing with this, as well as transgenic Cells, tissues, parts and reproductive material of such plants. Particularly preferred are transgenic crop plants, such as z. B. barley, wheat, rye, oats, corn, soy, rice, tree wool, sugar beet, rapeseed and canola, sunflower, flax, hemp, Potato, tobacco, tomato, rapeseed, tapioca, manioc, arrowroot, Alfalfa, lettuce and the various tree, nut and wine species. Plants in the context of the invention are monocotyledonous and dicotyledonous plants or algae.

Another embodiment according to the invention are as above described transgenic plants that have a functional or non-functional nucleic acid sequence according to the invention or a functional or non-functional according to the invention Expression cassette included. Under is not functional to understand that no more enzymatically active protein syn is thrown out. In addition, nucleus is not functional acids or nucleic acid constructs also a so-called anti Understand sense DNA that leads to transgenic plants that a reduction in enzymatic activity or no enzy have matic activity. With the help of antisense technology, especially if the nucleic acid sequence according to the invention with others Fatty acid synthesis genes combined in the antisense DNA is it possible triglycerides with an increased content of saturated To synthesize fatty acids or saturated fatty acids. Under transgenic plants are individual plant cells and their Cultures on solid media or in liquid culture, parts of plants and to understand whole plants.

Further objects of the invention are:

• - Method for transforming a plant thereby identified draws that expression cassettes containing a Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase gene sequence or DNA 'sequences hybridizing with this in a plant cell, in callus tissue, a whole plant or Introduces protoplasts from plants.
• - Use of a Δ6-acetylenase / Δ6-desaturase and / or Δ6-desaturase DNA gene sequence or hybridizing therewith DNA sequences for the production of plants with increased Content of fatty acids, oils or lipids with triple bonds or delta-6 double bonds by expression this D6 acetylenase / desaturase DNA sequence in plants.
• - Protein containing the amino shown in SEQ ID NO: 8 acid sequence.
• - Protein containing the amino shown in SEQ ID NO: 10 acid sequence.
• - Use of the proteins with the sequences SEQ ID NO: 8 and SEQ ID NO: 10 for the production of unsaturated fatty acids.

Another object of the invention is a method for Production of unsaturated fatty acids, characterized in that you have at least one of the above-described inventive Nucleic acid sequence or at least one according to the invention Nucleic acid construct into a preferably oil-producing one Organism brings, this organism attracts and that in that Organism contained oil isolated and those contained in the oil Releases fatty acids. These contain unsaturated fatty acids advantageously Δ6 triple and / or Δ6 double bonds. The fat acids can be extracted from the oils or lipids, for example via a basic hydrolysis e.g. B. be released with NaOH or KOH.

Also a method of making triglycerides with a increased content of unsaturated fatty acids, thereby marked draws that you have at least one fiction described above appropriate nucleic acid sequence or at least one according to the invention Brings the expression cassette into an oil-producing organism, attracts this organism and that oil contained in the organism isolated, belongs to the subject matter of the invention.

Another object of the invention is a method for Production of triglycerides with an increased content of unge saturated fat by taking triglycerides with saturated or unsaturated or saturated and unsaturated fatty acids with at least one of the protein represented by one of the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11 is encoded, incubated. That will be beneficial Process carried out in the presence of compounds which Absorb or release reduction equivalents. Afterward the fatty acids can be released from the triglycerides.

Method according to claim 16 or 17, characterized in that the fatty acids are released from the triglycerides.

The above-mentioned processes advantageously enable the synthesis of fatty acids or triglycerides with an increased content of Fatty acids with Δ6 triple and / or Δ6 double bonds.

With the help of so-called antisense technology, in one Process also fatty acids or triglycerides with an increased Saturated fat content.

Organisms for the processes mentioned are exemplary Plants such as Arabidopsis, barley, wheat, rye, oats, corn, Soy, rice, cotton, sugar beet, rapeseed and canola, suntan flower, flax, hemp, potato, tobacco, tomato, rapeseed, tapioca, Cassava, arrowroot, alfalfa, peanut, castor oil, coconut, oil palm, Safflower (Carthamus tinctorius) or cocoa bean, micro organisms such as fungi Mortierella, Saprolegnia or Pythium, Bacteria such as the genus Escherichia, cyanobacteria, yeasts like the genus Saccharomyces, algae or protozoa like Dinoflagellates called as Crypthecodinium. To be favoured Organisms that naturally syn oil in large quantities like microorganisms like fungi like Mortierella alpina, Pythium insidiosum or plants such as soy, rapeseed, coconut nut, oil palm, safflower, castor, calendula, peanut, cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae, Soy, rapeseed, sunflower and carthamus are particularly preferred or Saccharomyces cerevisiae.

The organisms used in the process will vary depending on the host organism attracted or ge in a manner known to the person skilled in the art breeds. Microorganisms are usually in a liquid Medium that contains a carbon source, usually in the form of sugars, a source of nitrogen mostly in the form of organic nitrogen swell like yeast extract or salts like ammonium sulfate, traces elements such as iron, manganese, and magnesium salts, if applicable Contains vitamins, at temperatures between 0 ° C and 100 ° C before attracted between 10 ° C to 60 ° C under oxygen gassing. The pH of the nutrient fluid can be set to a fixed value be maintained, i.e. regulated during cultivation or not. The cultivation can be batch-wise, semi-batch-wise or take place continuously. Nutrients can be used at the beginning of the Fermentation submitted or semi-continuous or continuous after being fed.

After transformation, plants are initially as described above regenerated and then grown as usual builds.

After cultivation, the lipids are obtained from the organisms in the usual way. For this purpose, the organisms can first be broken down after harvest or used directly. The lipids are advantageously extracted with suitable solvents such as non-polar solvents such as hexane or ethanol, isopropanol or mixtures such as hexane / isopropanol, phenol / chloroform / isoamyl alcohol at temperatures between 0 ° C and 80 ° C, preferably between 20 ° C and 50 ° C. The biomass is usually extracted with an excess of solvent, for example an excess of solvent to biomass of 1: 4. The solvent is then removed, for example by distillation. The extraction can also be carried out _{with supercritical CO 2.} After extraction, the remaining biomass can be removed, for example by filtration.

The crude oil obtained in this way can then be further purified be, for example, in the cloudiness over the offset with polar solvents such as acetone or chloroform and subsequent filtration or centrifugation can be removed. Further column cleaning is also possible.

To be used to extract the free fatty acids from the triglycerides these in usually saponified.

The invention also relates to unsaturated fats acids and triglycerides with an increased content of unge saturated fat obtained by the above procedure as well as their use for the production of Food, animal feed, cosmetics or pharmaceuticals. For this these become the food, the animal feed, the cosmetics or pharmaceuticals added in customary amounts.

The invention is illustrated in more detail by the following examples:

Examples example 1 General cloning procedures

The cloning methods such. B. restriction cleavages, Agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids on nitrocellulose and nylon membranes, ver linkage of DNA fragments, transformation of Escherichia coli Cells, cultivation of bacteria and the sequence analysis recombi As in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) described by guided.

Example 2 Sequence analysis of recombinant DNA

The sequencing of recombinant DNA molecules was carried out with a Laser fluorescence DNA sequencer from ABI using the method von Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467). Fragments resulting from a polymerase chain reaction were used to avoid polymerase errors in too express ming constructs are sequenced and checked.

Example 3 Generation of transgenic oilseed rape plants (modified from Moloney et al., 1992, Plant Cell Reports, 8: 238-242)

Binary vectors in Agrobacterium tumefaciens C58C1: pGV2260 or Escherichia coli were used to generate transgenic oilseed rape plants (Deblaere et al. 1984, Nucl. Acids. Res. 13, 4777-4788). For the transformation of oilseed rape plants (Var. Drakkar, NPZ Nor deutsche Pflanzenzucht, Hohenlieth, Germany), a 1:50 dilution of an overnight culture of a positively transformed agrobacterial colony in Murashige-Skoog medium (Murashige and Skoog 1962 Physiol. Plant. 15, 473) was used 3% sucrose (3MS medium) used. Petioles or hypocotyledons of freshly germinated sterile oilseed rape plants (approx. 1 cm ² each) were incubated in a Petri dish with a 1:50 agrobacterial dilution for 5-10 minutes. This is followed by a 3-day concubation in the dark at 25 ° C. on 3MS medium with 0.8% Bacto agar. The cultivation was continued after 3 days with 16 hours of light / 8 hours of darkness and in a weekly rhythm on MS medium with 500 mg / l Claforan (cefotaxime sodium), 50 mg / l Kanamycin, 20 microM benzylaminopurine (BAP) and 1 , 6 g / l glucose continued. Growing shoots were transferred to MS medium with 2% sucrose, 250 mg / l Claforan and 0.8% Bacto agar. If no roots formed after three weeks, 2-indolebutyric acid was added to the medium as a growth hormone for rooting.

Example 4 Generation of transgenic Arabidopsis thaliana plants

The transformation of Arabidopsis thaliana Var. Columbia Col 0 (Lehle Seeds, Round Rock, Texas, USA) was carried out using flowers infiltration method as described by: Bechtold, N., Ellis, J. and Pelletier, G. in Planta, Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants, C.R. Acad. Sci. Paris, Life Sciences 316: 1194-119 (1993) or by means of Root transformation method.

Example 5

The transformation of maize plants was carried out as in Pareddy, D., Petolino, J., Skokut, T., Hopkins, N., Miller, M., Welter, M., Smith, K., Clayton, D., Pescitelli, S., Gould, A., Maize Trans formation via helium blasting. Maydica. 42 (2): 143-154, 1997, described.

Example 6 Isolation and cloning of Δ6-acetylenase / Δ6-desaturase and Δ6-desaturase from Ceratodon purpureus

In order to isolate DNA sequences from Ceratodon purpureus which code for a Δ6-acetylenase / Δ6-desaturase and a Δ6-desaturase, various degenerate oligonucleotide primers were derived from DNA sequences which are for Δ5 (EMBL accession no. Z81122) and Δ6-fatty acid desaturases (U79010, AJ222980, AF031477 code:
Primer A: 5'-TGG TGG AA (A / G) TGG A (A / C) I CA (C / T) AA-3 ', forward primer, derived from the amino acid sequence WWKW (N / T / K) H ( N / K)
Primer B: 5 '- (T / G) GI TGG AA (A / G) (T / G) (G / A) I (A / C) AI CA (C / T) AA-3', forward primer, derived from the amino acid sequence (G / W) WK (E / D / W) (N / Q / K) H (N / K)
Primer C: 5'-AT (A / T / G / C) T (T / G) (A / T / G / C) GG (A / G) AA (A / T / G / C) A (A. / G) (A / G) TG (A / G) TG -3 ', reverse primer, derived from the amino acid sequence (I / M) (H / Q / N) PF (L / F) HH

Polymerase chain reaction (PCR) with single-stranded cDNA from C. purpureus was used to create two DNA fragments 557 bp (Cer3) and 575 bp (Cer16) in length with primer A and primer C and a DNA fragment of 560 with primer B and primer C bp (Cer1) length amplified. The following program was used for the amplification: 10 min at 94 ° C., pause for "hot start" at 72 ° C., followed by 32 cycles of 20 s at 94 ° C., 1 min at 45 ° C. (binding temperature, T _m ) and 1 min at 72 ° C, 1 cycle of 10 min at 72 ° C and stop at 4 ° C. Taq DNA polymerase (Gibco BRL) was used for the amplification.

The above double-stranded DNA fragments from the two PCR amplifications were in the pGEM-T vector (Promega) alloyed, transformed into E. coli XLlblue MRF 'Kan (Stratagene) and with the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt) sequenced. The DNA Partial sequences of Cer1 and Cer3 showed 70% identity. The above DNA partial sequences mentioned encoded without primer for open Reading frame for Cer1 of 173 amino acids (SEQ ID NO: 5 = partial Nucleotide sequence without primer from Cer1 and SEQ ID NO: 6 = Partial deduced amino acid sequence of Cer1), at Cer 3 of 172 amino acids (SEQ ID NO: 7 = partial nucleotide sequence without Primer from Cer3 and SEQ ID NO: 8 = Partially deduced amino acid sequence of Cer3) and for Cer16 of 178 amino acids (SEQ ID NO: 9 = partial nucleotide sequence without primer from Cer16 and SEQ ID NO: 10 = Partial deduced amino acid sequence of Cer16). The deduced protein sequence of Cer1 pointed 64% to Cer3 and 28% identical amino acids to Cer16; Cer 3 and Cer 16 again had 27% identical amino acids.

The Cer1 and Cer3 proteins are most similar on the Δ6-acyl lipid desaturase from Physcomitrella patens (Girke et al., Plant J., 15, 1998: 39-48), while Cer16 the highest similarity to the Δ6-acyl lipid desaturase and the Having Δ8-sphingolipid desaturase from higher plants.

A directed λZAP cDNA library from Ceratodon purpureus was made available by Fritz Thummler, Botanical Institute of the University of Munich (Pasentsis et al., Plant J., 13, 1, 1998: 51-61). A PCR test of this Ceratodon library was carried out in which specific primers were derived from the above-mentioned DNA partial sequences Cer1, Cer3 and Cer16:
Specific forward and reverse primers:

A restriction analysis (Hind III or EcoR V) of the cDNA Bank PCR amplified products showed in all three Create the same restriction pattern as that of the PCR amplicons from the ss-cDNA, d. H. the Ceratodon cDNA library contains the three Clones Cer1, Cer3 and Cer16.

Example 7 cDNA bank screening and sequencing of the "full length" clones

DNA mini-preparations of the three PCR amplified from ss-cDNA Fragments Cer1, Cer3, Cer16 ~ 570 bp in length in pGEM-T (see Example 6) were used for further screening of the complete Clones from a λ ZAP cDNA library from Ceratodon purpureus to M. Lee and S. Stymne delivered. This cDNA bank screening has so far been successful to two complete clones of Cer1 and Cer3 with inserts from approx. 2.2 kb, which as EcoR I / Kpn I fragments from the λ ZAP- Vector into the EcoR I / Kpn I sites of the puc19 vector (New England Biolabs) and subcloned into E. coli JM105 trans were formed.

Another screening of the cDNA library with Cer1 and Cer3 as Hybridization probes under low stringency indicated that at least one other Cer1-homologous clone exists which could possibly code for the Δ5-desaturase.

Two E. coli clones, Cer1-50 and Cer3-50, were completely sequenced. Cer1-50 has a length of 2003 bp (SEQ ID NO: 1 = nucleotide sequence of the Δ6-acetylenase / Δ6-desaturase from Ceratodon purpureus with 5 'and 3' untranslated regions and polyA) and codes for an open reading frame of 483 amino acids (SEQ ID NO: 2 = deduced amino acid sequence of the Δ6-acetylenase / Δ6-desaturase from Ceratodon purpureus). Cer3-50 has a length of 2142 bp (SEQ ID NO: 11 = nucleotide sequence [2142 bp] of the Δ6-desaturase from Ceratodon purpureus with 5 'and 3' untranslated regions) with an open reading frame of 520 amino acids (SEQ ID NO: 12 = deduced amino acid sequence of the Δ6-desaturase from Ceratodon purpureus). Both protein sequences have the highly conserved HPGG motif of cytochrome b ₅ at the N-terminal (Lederer F., Biochimie 76, 1994: 674-692) and the three histidine boxes characteristic of desaturases at the C-terminal (Shanklin et al., Biochemistry , 33, 1994: 12787-12794). They thus represent further members of the growing family of cytochrome b ₅ fusion proteins (Napier et al., Trends in PLant Science, 4, 1, 1999: 2-4). The first histidine in the third box has been replaced by glutamine, a further characteristic of Δ5- and Δ6-acyl lipid desaturases and Δ8-sphingolipid desaturases.

Example 8 Cloning of the entire functional active Δ6-acetylenase / Δ6- Desaturase and Δ6 desaturase sequence using PCR and the Providing this sequence for cloning in vectors as well as functional expression in yeast

A cDNA was prepared for enzymes with Δ6-acetylene ase / Δ6-desaturase activity from Ceratodon purpureus. Ana The Δ6-desaturase was log to the example described here cloned (see SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 12).

For this purpose, the oligonucleotides for a polymerase chain reaction (PCR) are first derived from the Cer1 cDNA for the Δ6-acetylenase / desaturase from Ceratodon purpureus.

The following primers were derived from Cer1 and adapted for expression in yeast:

In a PCR reaction, a Δ6-acetylenase / desaturase cDNA from Ceratodon purpureus is used as a template. Using the primers, a BamHI restriction site is introduced in front of the start codon of the Δ6-acetylenase / desaturase cDNA. For directed cloning, a SalI restriction site is introduced behind the stop codon. The reaction mixtures contained approx. 1 ng / micro l template DNA, 0.5 microM of the oligonucleotides and, 200 microM deoxy nucleotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C, 1.5 mM MgCl ₂ ) and 0.02 U / micro l Pwo polymerase (Boehringer Mannheim) and are incubated in a PCR machine from Perkin Elmer with the following temperature program:

Aging temperature: 50 ° C, 52 sec Denaturation temperature: 95 ° C, 52 sec Elongation temperature: 72 ° C, 90 sec Number of cycles: 30th

The resulting fragment of 1467 base pairs is in the with EcoRV cleaved vector pBluescript SK- (Stratagene) ligated. By Control cleavage, a clone is identified pBS-Cer1, whose Insert can be excised in full length by BamHI / SalI (1452 Base pairs plus 15 nucleotide restriction sites) and has the following sequence (the start and stop codon is below underlined, the interfaces are shown in italics). Analogue a cDNA sequence of the clone Cer50 can also be used. This is a monofunctional delta-6 desaturase (see SEQ ID NO: 3). The deduced amino acid sequence is SEQ ID NO: 4 can be found.

To check the functionality of the encoded enzyme in one Microorganism, the 1467 bp BamHI / SalI fragment from pBS Cer1 cut into the BamHI / XhoI expression vector pYES2 (Invitrogen, Groningen, Netherlands) ligated and yeast after Standard protocols with the newly created plasmid pYES2-Cer1 transformed (see transformation protocol Invitrogen, Groningen, Netherlands). Preserved colonies are based on raffinose medium containing attracted and using galactose the gene expression the Δ6-acetylenase / desaturase induced (see below).

Example 9 Lipid analysis of transformed yeast

In addition to their own fatty acids (16: 0, 16: 1, 18: 0 and 18: 1), yeasts can also incorporate exogenous fatty acids into their membrane lipids. To test the substrate specificity of the respectively expressed desaturase, 1% Tergitol NP-40 (w / v, Sigma) and 0.003% of the corresponding fatty acid (stock solution: 0.3% or 3% fatty acid in 5% Tergitol NP-40, w / v) added before inoculation. The preculture was carried out by inoculating 3 ml CM - 2% raffinose medium / 1% Tergitol NP-40 with a transgenic yeast colony and subsequent incubation for 2 days at 30 ° C in a roller up to an optical density of 600 nm (OD ₆₀₀ ) from 4.0 to 4.3. For the main culture, 10 ml CM - 2% raffinose / 1% Tergitol NP-40 medium ± 0.003% fatty acid are _{inoculated with an aliquot of the preculture (200-fold dilution) to an OD 600} 0.02 and 24 h at 30 ° C, 250 rpm incubated in the shaker. The induction of the test cultures took place in the logarithmic growth phase (OD ₆₀₀ 0.5 to 0.6) by adding galactose to 1.8%. The induced cells were harvested after a further 24 hours of aerobic growth at 30 ° C. with an OD ₆₀₀ of 4.0 to 4.3.

The induced yeast cells are centrifuged at for 10 min 2000 g harvested, in 3 ml distilled water. resuspended, 10 min at 100 ° C boiled and after cooling on ice, sedimented again. The cell sediment is treated with 1 N methanolic sulfuric acid and 2% dimethoxypropane hydrolyzed for 1 h at 90 ° C and the lipids trans methylated. The resulting fatty acid methyl esters (FAME) will be extracted in petroleum ether. The extracted FAME are through Gas-liquid chromatography with a capillary column (chromium pack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and one Temperature gradients from 170 ° C to 240 ° C in 20 min and 5 min 240 ° C analyzed. The identity of the mono-, di-, tri- and tetraene acid methyl ester is determined by comparison with the corresponding FAME Standards (Sigma) confirmed. For the tri- and tetrayn acids no reference substances are available. Your identity and the position of the triple bond is determined by appropriate chemical Derivatization of the FAME mixtures e.g. B. to 4,4-dimethoxyoxazoline Derivatives (Christie, 1998) analyzed by means of GC-MS from. The GC Analyzes of the fatty acid methyl esters from the transgenic yeasts that with the empty vector pYES2, with pYES2-Cer1 (Δ6-acetylenase) are transformed, is shown in Tab. The transgenic Yeast cells are made without exogenous fatty acids or after being added of linoleic acid (18: 2), γ-linolenic acid (γ-18: 3), α-linolenic acid (α-18: 3) or ω3-octadecatetraenoic acid (18: 4) analyzed.

Table 1 gives the GC analyzes of the fatty acid methyl esters from transgenic yeasts, which with the empty vector pYES2, the Δ6-acetylenase (Cer1 / pYES2) and the Δ6-desaturase (Cer3 / pYES2) have been transformed, again. The transgenic yeast cells were without exogenous fatty acids (-) or after adding linoleic acid acid (18: 2), γ-linolenic acid (γ-18: 3), α-linolenic acid (α-18: 3) or ω3-octadecatetraenoic acid (18: 4) analyzed. Fatty acid composition in [mol%] of total fatty acids, where the Incorporation of the fed fatty acids (black bold print), the desaturation products (in red) and the sum the desaturation products (last line) for the individual Feeding trials are indicated.

Example 10 Generation of transgenic plants which use an enzyme Overexpress D6 acetylenase / desaturase activity

For the transformation of plants, a transformation vector is generated which the BamHI / SalI fragment from pBS-Cer1 is ligated into the vector pBin-USP which has been cleaved with BamHI / SalI or into pBinAR. pBin-USP and pBinAR are derivatives of the plasmid pBin19. pBinAR arose from pBin19 by adding a 35S CaMV promoter as an EcoRI-KpnI fragment (corresponding to nucleotides 6909-7437 of the Cauliflower mosaic virus (Franck et al. (1980) Cell 21, 285) The polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., (1984) EMBO J. 3, 835), nucleotides 11749-11939 is isolated as PvuII-HindIII fragment and cloned after addition of SphI linkers to the PvuII cleavage site between the SpHI-HindIII cleavage site of the vector. The result was the plasmid pBinAR (Höfgen and Willmitzer (1990) Plant Science 66, 221-230), several restriction cleavage sites are available between promoter and terminator by recloning from pBluescript. The USP promoter corresponds to nucleotides 1-684 (Genbank Accession X56240), part of the non-coding region of the USP gene being contained in the promoter Base pair sized promoter fragment was created using commercially available T7 standard primer (Stratagene) and amplified with the aid of a synthesized primer via a PCR reaction according to standard methods. (Primer sequence:
5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCCGGATCTGCTGGCTATGAA-3 '). The PCR fragment was recut with EcoRI / SalI and inserted into the vector pBinAR. The result is the plasmid named pBinUSP.

The construct is used to transform Arabidopsis thaliana and rapeseed plants are used.

Regenerated shoots are grown on 2MS medium with kanamycin and Claforan received, transferred to soil after rooting and after Cultivation for two weeks in a climatic chamber or in Greenhouse grown, bloomed, ripe seeds harvested and for D6 acetylenase / desaturase expression using lipid analyzes examined. Lines with increased levels of acetylene fatty acids or double bonds at the delta-6 position identified. It can be converted into the stably transformed trans gene lines that functionally express the transgene increased content of acetylene fatty acids and double bonds the delta-6 position compared to untransformed control identify plants.

Example 11 Lipid extraction from seeds

The analysis of lipids from plant seeds is analogous to the analysis of yeast lipids. However, plant material is first mechanically homogenized using a mortar in order to make it accessible for extraction.

SEQUENCE LOG

Claims (23)

1. Isolated nucleic acid sequence which codes for a polypeptide with Δ6-acetylenase and / or Δ-6-desaturase activity, selected from the group:

• a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11,
• b) nucleic acid sequences which, as a result of the degenerate genetic code, are derived from the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11,
• c) Derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11, which code for polypeptides with the amino acid sequences shown in SEQ ID NO: 2 and have at least 75% homology at the amino acid level, without that the enzymatic effect of the polypeptides is significantly reduced.

2. Amino acid sequence encoded by a nucleic acid sequence according to claim 1. 3. Amino acid sequence according to claim 2, encoded by the in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 11 Sequence. 4. Expression cassette containing a nucleic acid sequence according to Claim 1, wherein the nucleic acid sequence with one or is linked to several regulation signals. 5. Vector containing a nucleic acid sequence according to claim 1 or an expression cassette according to claim 4. 6. Organism containing at least one nucleic acid sequence according to claim 1 or at least one expression cassette according to Claim 4 or at least one vector according to Claim 5. 7. The organism of claim 6, wherein the organism is is a plant, a microorganism or an animal. 8. Transgenic plant containing or not functional functional nucleic acid sequence according to claim 1 or a functional or non-functional expression cassette according to claim 4. 9. Process for the production of unsaturated fatty acids characterized in that at least one nucleic acid sequence according to claim 1 or at least one expression Cassette according to claim 4 in an oil producing one Organism brings, this organism attracts and that in that Organism contained oil isolated and those contained in the oil Releases fatty acids. 10. Process for the preparation of triglycerides with a increased content of unsaturated fatty acids, thereby marked draws that at least one nucleic acid sequence according to Claim 1 or at least one expression cassette according to Brings claim 4 into an oil-producing organism, attracts this organism and that ent holding oil isolated. 11. The method according to claim 9 or 10, characterized in that that the unsaturated fatty acids have an increased content unsaturated fatty acids with a triple bond or with a double bond in the Δ-6 position or a triple have bond and double bond in the Δ-6 position. 12. The method according to claims 9 to 11, characterized net that the organism is a plant or a Microorganism. 13. Protein containing the amino shown in SEQ ID NO: 8 acid sequence. 14. Protein containing the amino shown in SEQ ID NO: 10 acid sequence. 15. Use of proteins according to claim 13 or 14 for Her position of unsaturated fatty acids. 16. Process for the preparation of triglycerides with a increased content of unsaturated fatty acids by getting Triglycerides with saturated or unsaturated or ge saturated and unsaturated fatty acids with at least one the proteins according to claim 2, 13 or 14 are incubated. 17. The method according to claim 16, characterized in that the Triglycerides produced in the presence of a compound who those who can absorb or give up reduction equivalents. 18. The method according to claim 16 or 17, characterized in that that the fatty acids are released from the triglycerides. 19. Unsaturated fatty acids produced by a process according to claim 9 or 18. 20. Triglycerides with an increased content of unsaturated fat acids produced by a process according to claim 10, 16 or 17. 21. Use of a nucleic acid sequence according to claim 1 or an expression cassette according to claim 4 for the production of transgenic plants. 22. Use of a nucleic acid sequence according to claim 1 or a fragment thereof for the isolation of a genomic Sequence via homology screening. 23. Use of unsaturated fatty acids according to claim 19 or triglycerides with an increased content of unsaturated according to claim 20 for the production of food, animal feed, cosmetics or pharmaceuticals.