DE4433307A1 - An isolated nucleic acid fragment and products derived from it - Google Patents

Abstract

The invention relates to DNA sequences which code vegetable acyl transferases and the use of these sequences to change the fatty acid spectrum of lipids.

Description

The invention relates to DNA sequences for plant acyltransferases encode, as well as the use of this sequence to change the fat acid spectrum of lipids.

Oils and fats (triacylglycerols) are found in the food sector and in oleo chemical-technical field varied use, however different quality requirements, d. H. of their fatty acid spectra are put. In the chemical sector, triacylglycerols are possible most homogeneous fatty acid spectra desirable, d. H. all three glycerol po sitions should be esterified with the same fatty acid if possible. Depending on Purpose are very different fatty acids, such as. B. Laurin, Oleic, ricinoleic or erucic acid, interesting.

Fewer by-products fall when using such pure raw materials to, so that the effort for cleaning and processing considerably reduced and thus the cost / benefit ratio improved. So soon with oils high, consistent quality and available in sufficient quantities there may be new sales markets and possible applications open that are currently not viable from an economic perspective.

To ensure the quality of vegetable oils meet the requirements of chemical indu The following ways are generally available: firstly, the Development of powerful crops from wild plants, their Sa menöle have homogeneous fatty acid spectra, such as. B. Certain Cup Heads or Tropaeolum species, on the other hand the standardization of the fatty acid spectra of the oils of existing crops, such as. B. Raps, Son nenblume or soybean through classic breeding or specifically through genetic engineering interventions. Because the domestication of wild plants very much is time consuming and usually changing a multitude under  effort, focus on a wide variety of characteristics worldwide on the modification of existing crops. It it is known that by transferring a gene into sense or antisense orientation significant changes in the fatty acid spectrum of rapeseed oil can be aimed. Furthermore, it is through classic breeding or succeeded through genetic engineering to verify the fatty acid spectrum standardize different seed oils with regard to oleic acid.

The standardization of the fatty acid spectrum with respect to other indu Strictly interesting fatty acids that have a chain length of more or more Lich have less than 18 carbon atoms and in the following as unusual fatty acids are referred to, such as B. Erucic acid (cis-13- Docosenic acid, 22: 1) or lauric acid (decanoic acid 12: 0) has so far been all not yet achieved. From fatty acid analysis data of the oils and enzymati Studies on oil synthesis show that the content of such Fatty acids in the oil are not limited only by the enzyme activities, which are involved in the synthesis of these fatty acids, but also by those that catalyze the incorporation of fatty acids into the oil. Especially the 1-acyl glycerin-3-phosphate acyltransferase, which incorporates fatty acids into the middle catalyzed by the glycerol position of the oil is critical. This enzyme namely, in the ripening seeds or fruits of crops, such as B. rape, soy or corn, a very pronounced specificity and selek activity for unsaturated C₁₈ fatty acids. But it is with the unusual Chen fatty acids are not active, especially if the sn-1 position is already with of such a fatty acid is esterified. The properties of 1-acylglyce rin-3-phosphate acyltransferase is the crucial barrier for an even occupation of all three glycerol positions with usual fatty acids.

This is probably the reason that despite intensive breeding programs failed, e.g. B. the erucic acid content in the fatty acid gene a mix of rapeseed oil that is naturally rich like that of other Brassicaceae of very long chain fatty acids has to be increased significantly over 60%. Therefore must erucic acid, which has been in the oleochemical-technical field for a long time is used, first of all it is costly to clean what their use possibilities z. B. in the plastics industry and thus their sales market considerably limited. In 1993 the market volume for erucic acid production was rapeseed oil (erucic acid content 45%) in Germany e.g. B. at about 28,000 t.  

It is expected that the market volume can increase tenfold, as soon as oils with erucic acid contents become available by approx. 90%.

The homogeneous occupation of all three glycerol positions with erucic acid, such as also with other unusual fatty acids, can be found in the oil of cultivated plants zen, such as B. rapeseed, made possible by genetic engineering methods, e.g. B. by transformation with a gene construct that for a 1-acylgly encoded cerin-3-phosphate acyltransferase with desired properties. Such genes are found e.g. B. in certain wild plants in which they expressed in a seed-specific manner. These genes code for acyltransfera sen, the unusual fatty acids on the middle glycerol position over wear, especially if the sn-1 position is already with such fat acid is esterified. Genes expressed for very long chain acyl group specific 1-acylglycerol-3-phosphate acyltransferases encode and are thus suitable for the fatty acid spectrum z. B. respect Standardizing erucic acid can be found in Limnanthes species.

Genes encoding 1-acylglycerol-3-phosphate acyltransferases have been developed isolated from Escherichia coli and Salmonella typhimurium, their amino acid sequences are 94% identical (Table 1). Furthermore, cDNAs were made Yeast and maize described an E. coli mutant that has a defect in their 1-acylglycerol-3-phosphate acyltransferase, complement. These cDNAs code for polypeptides whose amino acid sequences are below each other and to those of the bacterial acyltransferases about 30% are table (Table 1). A clear proof that these cDNAs for 1- Encoding acylglycerol-3-phosphate acyltransferases, however, still stands out.

It is an object of the invention to provide a nucleic acid fragment with which the quality of vegetable oils and fats can be changed by expression in vegetable systems in such a way that their usability as oleochemical raw materials is improved and expanded. This task is solved with a nucleic acid fragment according to claim 1. The sequence of such a nucleic acid fragment is shown in Fig. 1.

A method has thus been invented, the fatty acid composition of Control lipids. Nucleic acid fragments necessary for a 1-acylglyce rin-3-phosphate acyltransferase (AGPAT) are provided to encode  to produce chimeric genes. These chimeric genes can be used to To transform plants or microorganisms and thus the fatty acid composition and fatty acid distribution of the glycerolipids and especially to change the triacylglycerols.

The invention relates to an isolated nucleic acid fragment which contains a DNA sequence which codes for a vegetable AGPAT or similar enzyme and whose amino acid sequence has an identity of at least 35 or more percent to the sequence isolated from Limnanthes douglasii and shown in Fig. 2 .

The isolated nucleic acid fragment is further characterized by that it isolated from a plant belonging to the class of Dicotyledoneae was that it was isolated from the group of the following plants: Lim nanthes, rapeseed, arabidopsis.

First, it is surprising that a ge from plants in this way won nucleic acid fragment with regard to the derived amino acid no significantly higher similarity to the only other Plants known sequence, namely that of maize, has as to the three other known sequences from bacteria and yeast, but that the similarity to yeast is even somewhat greater than that to maize (see Tabel le 2).

Secondly, it is surprising that we are using the method used were able to obtain a cDNA clone that from plants, ie a eukaryote, which originates and codes for an AGPAT that is attached to the memory Lipid synthesis is involved and specific for very long chain fatty acids is because the mutant used for the complementation step is a Bak terium, i.e. a prokaryote, is the defect in membrane biosynthesis is present.

The nucleic acid fragment isolated from Limnanthes douglasii ( Fig. 1) is characterized in that the associated genes are expressed in a seed-specific manner and in that it codes for an AGPAT which is specific for very long-chain acyl groups - hereinafter this enzyme is referred to as L-AGPAT.

The isolated nucleic acid fragment is primarily characterized by  that it can be used to trans trierucine in the oil gener plants and thus increase its technical usability improve. Trieruci has the scientific name Trierucoylglycerin.

The invention further relates to the use of the nucleic acid question to isolate genes or cDNAs used for other plant Encode acyltransferases, e.g. B. Use of the nucleic acid fragment or parts thereof as a hybridization sample using from the Se sequence of the nucleic acid fragment derived oligonucleotides in the Po lymerase chain reaction (PCR), and use of antibodies against a Polypeptide derived from the nucleic acid fragment or its derivatives is encoded.

The invention also relates to all plasmids, viruses and other vectors which contain the isolated nucleic acid fragment or parts thereof, and all organisms, in particular plants and parts of plants which contain these constructs, and all products which are produced in the transgenic organisms and their material Composition is changed by the effect of the above sequences or parts thereof. Of particular interest are those plasmids (chimeric genes) that enable transcription or transcription and translation (expression) of plant acyltransferases in host cells, especially plant cells. These chimeric genes comprise nucleic acid fragments which code for a plant AGPAT or a similar enzyme and whose amino acid sequences have an identity of at least 35 or more percent to the sequence given in FIG. 2, as well as suitable control sequences which functionally function with the fragment are connected, which in turn can be inserted in "Sense" or "Antisense". Protection claims are also made on microorganisms, cell cultures, plants and parts of plants which contain these chimeric genes, and on products obtained therefrom, in particular triacylglycerol, the composition of which is specifically modified by the action of these chimeric genes.

The invention also relates to a method for the production of lipids which changed levels or distributions of very long chain fat acids, especially erucic acid.

This method involves the following steps:

• (a) Transformation of cells with a chimeric gene as described above  ben,
• (b) isolation of transgenic cell lines (clones) as described under (a),
• (c) Selection of cell lines from (b) whose lipids contain the desired fatty acid exhibit remuster,
• (d) Processing the cells from (c) to lipids with desired fatty acid to receive patterns.

Preferred cells are those from the following group: E. coli, Saccharo myces, plant cells. The cell lines and products made using this Method are also the subject of the invention.

The invention also relates to a method for the production of vegetable oils and fats, the changed contents or changed distributions for a very long time chain fatty acids, especially erucic acid, in their fatty acid spectra point. The production of plants is of particular interest Oils and fats with changed levels of trierucine.

This method involves the following steps:

• (a) Transformation of a plant cell with a chimeric gene as above described
• (b) growing fertile plants from cells as described under (a),
• (c) selection of progeny seeds from (b), the seed oils of which have the desired fatty acid pattern, and
• (d) processing the seeds from (c) to oil with desired fatty acid get star.

Plant cells from oil plants, such as. B. rapeseed, sunflower or flax prefers. Preferred methods for plant cell transformation are indi right DNA transfer with Ti and Ri plasmids from Agrobacterium, more direct DNA transfer, electroporation or ballistic DNA transfer.

The invention also relates to the transgenic plants and parts of plants which were produced with the help of this method, and those from the transgenic Plants or parts of plants obtained oils and fats with modified Fatty acid spectra.

The invention also includes a method of plant breeding to to maintain the changed quality of oils and fats from oilseeds. The method involves the following steps:

• (a) crossing the above transgenic plants with different, different Va advice,  
• (b) Southern blot hybridizations of restriction endonucleases digested genomic DNA from (a) with labeled nucleic acid fragments which are suitable for encode the claimed AGPATs.

The invention also relates to all manufactured using this method Plants and their descendants, and parts thereof and those made from them Products, especially triacylglycerols, as far as their material combination change due to the effect of the inserted nucleic acid fragments is.

Finally, the invention includes a method to isolate further nucleic acid fragments that encode AGPAT and related enzymes. It includes the following steps:
Comparison of the sequence in Fig. 2 with other acyltransferase amino acid sequences;
Identification of conserved areas (a); and
Production of region-specific oligonucleotide probes in order to produce the above-mentioned nucleic acid fragments using sequence-dependent protocols.

The nucleic acid fragments produced using this method as well Parts of this are also part of the invention, all chimae alike genes containing these fragments or parts thereof are all transgenic Microorganisms, cell lines and transgenic plants and parts thereof which o. g. Contain nucleic acid fragments and finally all products, which are made from this transgenic material and their material Composition by the action of the inserted nucleic acid fragments is changed.

The figures serve to explain the present invention. she demonstrate:

Fig. 1. DNA sequence of the pCH21 nucleic acid fragment isolated from a cDNA expression gene bank of developing Limnanthes douglasii embryos. It has a length of 1020 base pairs (bp) plus a PolyA sequence of 19 bp, a GC content of 41.9% and contains an open reading frame from positions 1 to 852. Start and stop codons are found at the positions 10 to 12 or 853 to 855.

Fig. 2. Amino acid sequence in the one-letter code, which was derived from the DNA sequence of the nucleic acid fragment pCH21 and which codes for an L-AGPAT with a molecular mass of 27.5 kDa.

Fig. 3. Sequence comparison of the 5′-region of the nucleic acid fragment pCH21 with that of another nucleic acid fragment pCH149 isolated from the cDNA expression gene bank of developing embryos of Limnanthes douglasii. The start codon (***) and the stop codon (+++) located upstream in the same reader are marked.

Fig. 4. Comparison of the amino acid sequences of the AGPAT of E. coli (line 1) and Salmonella typhimurium (line 2), as well as that derived from the cDNAs of yeast (line 3) and that of maize (line 4) with that in Fig. 2 specified. "*" means identical amino acids in all sequences, "·" means similar amino acids in all sequences. The comparison was carried out with the aid of the CLUSTAL program (Higgins & Sharp 1988, Gene 73) 237-244).

Fig. 5. Evidence of seed-specific expression by Northern blot analysis. Electrophoretically separated mRNA from leaves (lane 1: 1 µg; lanes 3 and 6: 3 µg and ripening seeds (lane 2: 1 µg; lanes 4 and 5: 3 µg) from Limnanthes douglasii was hybridized, the range from Nu kleotid 10 to nucleotide 741 of the nucleic acid fragment pCH21 shown in FIG. 1 was used as a radioactively labeled sample.

Table 1: Comparison of the amino acid identities of the known AGPATs. It mean ECO Escherichia coli, SAL Salmonella thyphimurium, SAC Saccha romyces cerevisiae and ZEA Zea mais. The values were determined using the Pro BESTFIT (Devereux et al 1984, Nuc. Acids Res. 12, 387-394).

Table 2: Comparison of the amino acid idenities of the known AGPATs. Be Drawing as in Table 1, LIM Limnanthes douglasii.

The invention is explained below using examples that all molecular biological methods such as DNA (plasmid) isolation, Agaro sailing electrophoresis, gel elution, phenolization, DNA fillings, ligation, Transformation, cloning, polymerase chain reaction (PCR) if not otherwise stated, according to standard protocols by Sambrook et al (Molecu lar Cloning, CSH Laboratory Press 1989).  

Example A Isolation of cDNAs for an L-AGPAT using heterologous complementation are listed 1. Preparation of PolyA + RNA from Limnanthes douglasii

To obtain PolyA + RNA, 4 g of Limnanthes douglasii embryos (approx. 10 days old) were finely ground under liquid nitrogen and mixed with 30 ml of extraction buffer (100 mM NaCl, 50 mM Tris pH 9.0, 10 mM EDTA, 2% ( w / v) SDS, 200 µg / ml proteinase K) added. After incubation for 10 minutes at 37 ° C., two phenol / chloroform (1: 1) extractions and one chloroform extraction followed with equal volumes. The phase separation was achieved by centrifugation at 6000 × g. After adding 1/5 vol 10 M LiCl₂ and incubating on ice for 1 hour, a centrifugation at 6500 × g, 4 ° C, was connected for 30 min and 0.2 g of Oli go-dT cellulose (Boehringer) was added to the supernatant. The cellulose was washed three times with washing buffer 1 (400 mM NaCl, 10 mM Tris pH 7.5, 0.2% (w / v) SDS) and washing buffer 2 (100 mM NaCl, 10 mM Tris pH 7.5) and the mRNA eluted with elution buffer (10 mM Tris pH 7.5). The eluted mRNA was purified a second time over oligo-dT cellulose as explained above. The yield was 20 µg RNA per 4 g fresh weight used.

2. Creation of a Limnanthes douglasii cDNA expression gene bank

For the isolation of cDNAs which are suitable for 1-acylglycerol-3-phosphate-acyltransfera encode, Limnanthes douglasii became a cDNA expression gene bank created in the phage vector Lambda-ZAP (Stratagene). For this, 2 µg mRNA used, which, as described under point 1, from maturing Embryos from Limanthes douglasii were isolated. The creation of the Genbank was carried out according to the manufacturer. The primary bank contains 3.7 × 10⁶ independent clones in a volume of 500 µl.

1 × 10⁶ clones (= 135 µl) of the primary bank were according to the manufacturer amplified information. This amplified library contains 2 x 1013 clones in 200 ml. 1 μl (= 108 clones) of which was transferred to phagemids, which in a A volume of 3 ml was available. With 5 µl Phaegemidsuspension (= 1.66 × 10⁵ Clones), bacterial cells were transfected and the resulting cells Plasmid DNA isolated. This was called the plasmid cDNA expression bank The preparation has a DNA concentration of 1 µg / µl.  

3. Heterologous complementation

The problem is the isolation of such clones, the AGPAT encoding cDNAs contain, from the mass of different clones, the other cDNAs of the Gene bank included. For this, the method of heterologous complements was used tation selected. The heterologous complementation is based on mutants resorted to, which have a defect in the enzyme, the associated rige cDNA is searched. The mutants used are usually con ditional. So there are permissive conditions under which growth of cells is possible and non-permissive conditions under which none Growth takes place. Now an entire gene bank becomes such a mutant transformed in the shotgun process and the transformed cells len under non-permissive conditions, then it can in growth in a few cases when the introduced cDNA encodes an AGPAT, this cDNA is sufficiently full is constant, the cloning in the correct orientation regarding the Expression promoter and in the correct reading frame and that hetero loge, Pro from the gene bank, that is from a foreign organism tein is sufficiently stable in the bacteria and in the foreign environment Exercise conditions is enzymatically active.

Such heterologous complementations have so far been in some cases has been carried out successfully, as documented in the literature.

In the present case, the mutant JC201 was used. This mutant has a defect in AGPAT that is thermosensitive. At 30 ° C NEN the cells grow, however, at 37 ° C cultivation temperature there is none Growth instead, AGPAT activity is no longer detectable in vitro, and 1-acylglycerol-3-phosphate accumulates in the mutant cells. Cells This mutant was identified by the method of Inoue et al (1990) in the State of competence transferred in which they can take up plasmid DNA nen. For the heterogeneous complementation, 200 µl each of the compe mutant cells with 100 ng plasmid DNA of the plasmid cDNA Expression library of Limnanthes douglasii transformed (Cohen et al 1972) and containing 100 µg / ml ampicillin under selective conditions LB plates incubated at 37 ° C. 300 clones were isolated at 37 ° C could grow. The plasmid DNA was isolated from these clones. The This DNA was then individually transformed into competent JC201, and the transformed cells were tested for their ability to not permissive temperature of 37 ° C to grow. This was with 130 clones  the case. The DNA belonging to these 130 clones was again compe tente JC201 transformed and the selection repeated. After this Step 27 still showed reproducible clones, the ability to at 37 ° C to grow. In another round, the number of positive ones decreased Clones on 9. This number then remained in three further selection rounds bil.

4. Analysis of the positive clone cDNA

The plasmid DNA isolated from the positive clones was analyzed using the Restriction analysis characterized in more detail. The plasmid DNA from six of the Clones (pCH21, pCH147, pCH148, pCH149, pCH170 and pCH186), their cDNA is about 1 kbp in length due to similar restriction patterns He grouped together and sequenced from both sides. It the cDNAs were found to contain identical nucleotide sequences and differ only in their 5'- and 3'-extent.

5. pCH21

The cDNA insert from pCH21 was completely sequenced on both strands. The nucleotide sequence is shown in Fig. 1. The cDNA contained has a length of 1039 bp. In the same orientation as the lacZ promoter, there is a long, open reading frame of 852 bp that is in the same phase as LacZ 'and therefore potentially forms a fusion protein. Including the stop codon, there are 279 base pairs in the 3′-untranslated region and a polyA region of 19 nucleotides. The nucleotide sequence has a GC content of 41.9%, which is typical for plant cDNAs.

The translation of eukaryotic mRNAs begins at the furthest 5 ′ located start codon (AUG = ATG in the cDNA). If one bases this on the first ATG occurring in the sequence of pCH21, the following sequence results in a potential polypeptide of 281 amino acids and a molecular weight of 27.5 kDa. The amino acid sequence is shown in Fig. 2 in the one-letter code

6. Completeness of the reading frame

Due to the production of cDNA clones, these are incomplete at the 5 'end permanent copies of the associated mRNA, the length of their 5'-ends vary. This is also the case for the six cDNAs mentioned above, which are identical in their sequence, as already stated. It  it can therefore be assumed with certainty that the cDNAs are copies of the mRNA of a gene.

So since pCH21 is not complete and also has an open reading frame in front of the first start codon at position 10, the question arises whether there is a further start codon further in the 5 ′ direction, so that the sequence shown in FIG Sequence that AGPAT represents. The sequence of pCH148 and pCH149, which is further extended in the 5′-direction, shows a stop codon in the 5′-direction in the reading frame under discussion ( Fig. 3). This limits the open reading frame in the 5 'direction, and it can be assumed with certainty that the start codon at position 10 in pCH21 represents the AGPAT start codon.

Example B Functional expression of 1-acylglycerol-3-phosphate Acyl transferase from Limnanthes douglasii in E. coli

To prove that the cDNA insert of pCH21 is a 1-acylgly cerin-3-phosphate acyltransferase, namely an Erucoyl-CoA-specific functional expression studies were carried out in E. coli leads and analyzed the acyl-CoA specificities of the expression product.

1. Construction of the expression plasmid pQEL21

The cDNA insert of pCH21 ( Fig. 1) was amplified using the polymerase chain reaction (PCR). The following two primers A and B, which have restriction sites for Nco I and Bgl II at their 54 ends, served as oligonucleotide primers ( FIG. 2).

For a 100 μl PCR batch, 10 ng pCH21, the primers A and B in 1 μM concentration, 5 units Taq DNA polymerase (Gibco) and 100 μM dNTPs in a buffer medium of 25 mM TAPS (pH 9.3), 2 mM MgC12, 50 mM KCl, 1 mM DTT, 0.5 mg / ml activated salmon sperm DNA was used. The PCR reaction was carried out in a thermal cycler (Biometra) at the following reaction temperatures and times: 2 min, 95 ° C for the first time denaturing the DNA, followed by 27 cycles with 20 sec each, 950 C for denaturing the DNA, 20 sec , 65 ° C for hybridization of the primers and 1 min, 72 ° C for amplification of the DNA. Then all DNA molecules were completely amplified for 3 min at 72 ° C. The 852 bp amplification product was separated from primers and by-products by agarose gel electrophoresis (Sambrook et al 1989), eluted from the gel using the QIAEX gel elution kit (QIA-GEN) according to the company's instructions and using the SUREclone kit (Pharmacia)) as by the manufacturer indicated, ligated into the dephosphorylated vector pUC19 linearized with Sma I. The plasmid pUCL21 thus obtained was checked by restriction and sequence analysis. Subsequently, 10 μg of the plasmid were digested with Nco I and Bgl II, and the Nco I / Bgl II fragment carrying the cDNA insert was isolated as described above by agarose gel electrophoresis and into the vector pQE60 (QIAGEN) linearized with Nco I and Bgl II. ligated to obtain the construct pQEL21. pQEL21 and the vector pQE60 were transformed into competent cells of the E. coli mutant JC201 which contained the plasmid pREP4 (QIAGEN) carrying the lac I ^q repressor.

2. Cultivation of the transgenic E. coli JC201 cells

The cultivation of E. coli JC201 cells, which in addition to the repressor plasmid the construct pQEL21 or the vector pQE60 contained was carried out in Ampi cillin (100 µg / l) and kanamycin (25 µg / l) containing LB medium at 30 ° C. Overnight cultures were diluted 1:40 in this medium and at 30 ° C tightened to an OD₆₀₀ of 0.6. For induction of the expression con Structured pQEL21 was isopropyl-β-D-thiogalactopyranoside (IPTG) in one Final concentration of 2 mM was added and the bacteria after a three hour incubation at 30 ° C by centrifugation at 4500 × g for 10 min harvested.

3. Membrane isolation from transgenic E. coli JC201 cells

All of the following steps were carried out at 0-4 ° C: By Sedimenta tion harvested bacterial cells were twice with membrane buffer (50 mM Tris / HCl pH 8.4, 5 mM MgCi2, 5 mM dithiotreitol) and washed in this Buffer resuspended so that the volume of the suspension is about 1/40 of Corresponded to the cultural volume. The bacterial cells were lysed by four times ultrasound treatment for 30 sec with the help of an ultrasound sta esp. After separation of large cell fragments and "inclusion bodies" by Centrifugation at 7500 × g for 10 minutes, the membranes were removed from the Cell homogenates sedimented by centrifugation at 10,000 × g for 1 h,  resuspended in a little membrane buffer and with glycerin in an end con concentration of 50% (v / v) stored at -20 ° C. The protein determination is successful using the Bradford method (Arial. Biochem. 72, 248-254 (1987)).

4. Enzyme test for determining the 1-acylglycerol-3-phosphate acyltransferase activity

The enzyme test is based on the enzymatic conversion of 1-acyl-sn- [U- ¹⁴C] glycerin-3-phosphate with acyl-CoA to 1,2-diacyl-sn- [U-¹⁴C] glycerin-3- phosphate. The reaction mixture contained 0.1 M tricin / NaOH pH 8.8, 4 to 40 µM oleoyl-CoA or erucoyl-CoA, 2.5 µM 1-oleoyl-sn- [U-¹⁴C] glycerol-3- phosphate (353 dpm / pmol) and membrane fractions (0.2-3.5 µg protein) in one em total volume of 50 µl. After a 15 minute incubation at 30 ° C was the batch with 50 ug phosphatidic acid in 240 ul chloroform: methanol (1: 1) and 100 ul 1 M KCl, 0.2 M H₃PO₄ mixed, mixed and phase separation centrifuged for 2 min at 1000 × g, 90 µl of the chloroform phase Ready-made silica gel plates applied and in chloroform: pyridine: formic acid (50: 30: 7) 30 min developed. The phosphatidic acid band was sprayed hen the developed silica gel finished plate with phosphatide reagent (Dittmer & Lester, J. Lipid Res. 5, 126-127 (1964)) localized in a scintillation scraped the tube and mixed with 5 ml cocktail (OptiPhase 4HISAFE4, Wallec) puts. The radioactivity of the sample was determined in the scintillation counter.

From these values, the specific enzyme activities were calculated as pmol of 1,2-diacylglycerol-3-phosphate per minute and mg of membrane protein. In enzyme tests with membrane fractions from pQE60-containing JC201 cells, neither oleoyl-CoA nor erucoyl-CoA reacted 1-acyl-sn- [U-¹ C] glycerol-3-phosphate to 1,2-diacyl-sn- [U-¹⁴C] glycerol-3-phosphate detectable. In contrast, the membrane fractions from pQEL21-containing JC201 cells catalyzed this reaction and showed higher specific enzyme activity with erucoyl-CoA than with oleoyl-CoA (200 pmol / min / mg protein with erucoyl-CoA and 84 pmol / min / mg protein with oleoyl -CoA). These results thus demonstrate that the cDNA insert of pCH21 for an erucoyl-CoA-specific 1-acylglycenn-3-phosphate acyltransferase codes for the acyltransferase which is expressed in Limnanthes in a seed-specific manner. These results are supported by Northern blot analyzes ( Fig. 5).

They show that the gene belonging to pCH21 is found in seeds but not in leaves tern is transcribed.  

Definitions

Plant parts are parts of a plant such as B. seeds, fruits or Fruit clusters.

Vectors are nucleic acid fragments that operate under certain conditions are capable of duplication and for the installation of other Nu small acid fragments for the purpose of multiplying this fragment or the Expression of this fragment (for example for the production of a protein) to be used. Typical examples are plasmids and phages.

Chimeric gene is a nucleic acid fragment that consists of different parts is composed and does not naturally occur in this form. It comprises a sequence coding for a polypeptide and suitable control sequences that enable expression. The coding sequence can with regard to the control sequences in "sense" or "antisense" orias available.

Very long chain fatty acids are those fatty acids that are a number of have more than 18 carbon atoms.

Vegetable oil is an oil that is used in pressing and / or extracting plant zen or parts of plants.

Isolation is the extraction of certain things from a mixture different things. Things can be substances (e.g. proteins, nu small acid fragments, mRNA, DNA, cDNA, cDNA clones, genes), cell parts (e.g. Membranes), cells (e.g. bacterial cells, plant cells), cell lines, organ men and their descendants.  

Table 1

Table 2

Claims (41)

1. An isolated nucleic acid fragment which contains a nucleic acid sequence which codes for the 1-acylglycerol-3-phosphate acyltransferase or similar enzymes and whose amino acid sequence has an identity of at least 35% to the sequence shown in FIG. 2. 2. An isolated nucleic acid fragment according to claim 1, wherein the identity of the associated amino acid sequence is at least 40%, preferably 45%, in particular 50% of the sequence shown in FIG. 2. 3. An isolated nucleic acid fragment according to claim 1, wherein the identity of the associated amino acid sequence is at least 65%, preferably at least 90%, of the sequence shown in FIG. 2. 4. An isolated nucleic acid fragment according to claim 1, characterized in that the amino acid sequence is the sequence according to FIG. 1. 5. An isolated nucleic acid fragment according to one of claims 1 to 4, characterized in that it is isolated from plants. 6. An isolated nucleic acid fragment according to one of claims 1 to 4, characterized in that it is isolated from dicotyledonous plants. 7. An isolated nucleic acid fragment according to one of claims 1 to 4,  characterized in that the starting material for isolation triacyl is glycerin synthesizing tissue. 8. An isolated nucleic acid fragment according to one of claims 1 to 4, characterized in that the starting material ripens for isolation Are seeds. 9. An isolated nucleic acid fragment according to one of claims 1 to 4, characterized in that the starting material ripens for isolation Limnanthes douglasii seeds are. 10. An isolated nucleic acid fragment according to one of claims 1 to 9, characterized in that it is expressed in a seed-specific manner. 11. plasmids, viruses or other vectors, characterized in that they Contain nucleic acids according to any one of claims 1 to 10. 12. Genomic clones, characterized in that they contain genes or parts of Genes included that correspond to a sequence or parts of the sequence encode one of claims 1 to 10. 13. A chimeric gene that is capable of converting the fatty acid composition into to transform a transformed cell and a nucleic acid fragment according to any one of claims 1 to 12, which in effective form with ge appropriate control sequences is linked. 14. A chimeric gene that is able to control the content of very long-chain, saturated and / or unsaturated fatty acids in a transformed cell le to change and a nucleic acid fragment according to any one of claims 1 contains to 12, which ver in effective form with suitable control sequences is knotting. 15. A chimeric gene capable of reducing the erucic acid content in a transformed cell, and a nucleic acid fragment according to any one of claims 1 to 12, which in effective form with ge appropriate control sequences is linked. 16. A chimeric gene that is able to modify the fatty acid composition in the  Change triacylglycerol in a transformed cell, and a nucleotide Contains acid fragment according to any one of claims 1 to 12, which is active in mer form is linked with suitable control sequences. 17. A chimeric gene that is capable of containing very long chain Change fatty acids in triacylglycerol in a transformed cell, and contains a nucleic acid fragment according to any one of claims 1 to 12, that is effectively linked to appropriate control sequences. 18. A chimeric gene that is able to reduce the level of erucic acid in the Change triacylglycerol in a transformed cell, and a nucleotide Contains acid fragment according to any one of claims 1 to 12, which is active in mer form is linked with suitable control sequences. 19. A chimeric gene according to claims 13-18, which is capable of Allow synthesis of trierozin in a transformed cell where the gene is a nucleic acid fragment according to any one of claims 1-12 contains, which is linked in an effective form with suitable control sequences is. 20. A chimeric gene according to any one of claims 13-19, thereby characterized in that the construction in "Sense" and / or "Antisense" -Ori entation is present. 21. Transformed cells that have a chimeric gene corresponding to one of the Claims 13 to 20 included. 22. Transformed cells according to claim 21, characterized net that they contain 1-acylglycerol-3-phosphate acyltransferases (AGPAT), those in these naturally not or in another form or other Concentration. 23. Transformed cells according to claim 21, characterized net that they contain 1-acylglycerol-3-phosphate acyltransferase activities ten, of course not in these or at a different level or with other specificity. 24. Transformed cells according to claim 21, characterized  net that they are specific for very long-chain acyl groups 1-acylglycerol-3- phosphate acyltransferase activities (L-AGPAT activities) that naturally do not occur in these or at a different level. 25. Transformed cells according to claim 21, characterized net that they are erucic acid specific 1-acylglycerol-3-phosphate acyltransfera contain seaactivities, which naturally do not exist in these or in other higher altitude. 26. Plants or parts of plants that have a chimeric gene corresponding to one of claims 13 to 20 included. 27. Plants or parts of plants according to claim 26, characterized records that they contain 1-acylglycerol-3-phosphate acyltransferases, which in these naturally not or in another form or in another con concentration occur. 28. Plants or parts of plants according to claim 26, characterized records that they are specific for very long chain acyl groups 1-acylglyc contain rin-3-phosphate acyltransferase activities (L-AGPAT activities), which naturally do not occur in these or at a different level. 29. Plants or parts of plants according to claim 26, characterized records that they erucic acid specific 1-acylglycerol-3-phosphate-acyl contain transferase activity, which naturally does not exist in these or in of a different height. 30. Plants or parts of plants according to claim 26, characterized records that they are seed-specific 1-acylglycerol-3-phosphate acyltransfera seaktivitdt included, which in these naturally not or in others Height. 31. Oil obtained from seeds or fruits of plants that are a chimeric Contain gene according to claims 13 to 20. 32. Oil from plants or parts of plants, which according to one of the An proverbs 26 to 30 is won.   33. A method for producing 1-acylglycerol-3-phosphate acyltransferase, which comprises the following items

• (a) transforming a cell with a chimeric gene according to any one of claims 13 to 20;
• (b) selection of those cells which produce the protein in the desired amount;
• (c) and preferably purifying the protein from other ingredients.

34. A method for producing plants and / or vegetable oil with an altered content of very long-chain fatty acids, which comprises the following points:

• (a) transforming a plant cell of an oil-producing plant with a chimeric gene according to any one of claims 13 to 20;
• (b) growing fertile plants from the transformed plant cell according to (a);
• (c) selection of the progeny of the plants in accordance with (b), in order to obtain the desired content of very long-chain fatty acids;
• (d) processing the seeds of the plants according to (c) in order to obtain plants and / or vegetable oil which has the desired content of very long-chain fatty acids.

35. The method according to claim 34, characterized in that the very long chain fatty acid is erucic acid. 36. A method for producing plants and / or vegetable oil with a modified trierucine content, which comprises the following points:

• (a) transforming a plant cell of an oil-producing plant with a chimeric gene according to any one of claims 13 to 20;
• (b) growing fertile plants from the transformed plant cell according to (a);
• (c) selection of the progeny of the plants in accordance with (b) in order to obtain the desired trierucine content;
• (d) processing the seeds of the plants according to (c) in order to obtain plants and / or vegetable oil which has the desired Trieru cin content.

37. The plant and / or the vegetable oil which according to one of claims 34- 36 is made.   38. A method of obtaining nucleic acid fragments encoding acyl transferases and acyl transferase-like enzymes, comprising the steps of:

• (a) Comparison of the amino acid sequence in FIG. 2 with the amino acid sequences of other acyltransferases;
• (b) identifying one or more conserved sequence regions with 4 or more identical amino acids as a result of (a);
• (c) production of specific oligonucleotides by back-translating the amino acid sequence according to (b);
• (d) Using these oligonucleotides to produce nucleic acids encoding acyl transferases or acyl transferase-like enzymes using a sequence-dependent protocol.

39. A method according to claim 38 to extract nucleic acid fragments from plant zen, especially dicotyledonous plants. 40. seed oil obtained from plants according to claim 38, thereby ge indicates that it contains trierucine. 41. A process to make seed oil with a modified trierucine content which comprises the following points according to claim 38 with (trierucine).