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WO1997037006A1 - Enzyme de plante et utilisation de cette derniere - Google Patents

Enzyme de plante et utilisation de cette derniere

Info

Publication number
WO1997037006A1
WO1997037006A1 PCT/SE1997/000554 SE9700554W WO9737006A1 WO 1997037006 A1 WO1997037006 A1 WO 1997037006A1 SE 9700554 W SE9700554 W SE 9700554W WO 9737006 A1 WO9737006 A1 WO 9737006A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
phospholipase
uncommon
organisms
fatty acid
Prior art date
Application number
PCT/SE1997/000554
Other languages
English (en)
Inventor
Sten Stymne
Ulf STÅHL
Bo Ek
Staffan Sjödahl
Original Assignee
Sten Stymne
Staahl Ulf
Bo Ek
Sjoedahl Staffan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sten Stymne, Staahl Ulf, Bo Ek, Sjoedahl Staffan filed Critical Sten Stymne
Priority to AU25256/97A priority Critical patent/AU2525697A/en
Priority to EP97916701A priority patent/EP0904358A1/fr
Publication of WO1997037006A1 publication Critical patent/WO1997037006A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention relates to use of a plant enzyme gene for transformation. More specifically, the invention relates to use of a previously not described phospholipid acyl hydrolase gene in combination with a gene for an uncommon fatty acid for obtaining transgenic plants comprising both said genes.
  • diacylglycerols is a common precursor for both phospholipids and triacylglycerols in plant tissues accumulating triacylglycerols (see Stymne, 1993a for review).
  • CDP-choline choline phosphotransferase in plant tissues accumulating high amounts of medium chain and hydroxy fatty acids in their triacylglycerols do not discriminate against diacylglycerols containing these fatty acids in the synthesis of phosphatidylcholine (Vogel & Browse, 1995).
  • unusual fatty acids of interest e.g. medium chain fatty acids, hydroxy fatty acids, epoxy fatty acids and acetylenic fatty acids
  • the present inventors have found that, in plant species naturally accumulating these uncommon fatty acids in their seed oil (triacylglycerols), these acids are absent, or present in very low amounts, in the membrane (phospho) lipids of the seed. The low concentration of these acids in the membrane lipids is most likely a prerequisite for proper membrane function and thereby for proper cell functions.
  • the idea underlying the invention is that uncommon fatty acids can be made to accumulate to high amounts in seeds of transgenic crops if these uncommon fatty acids are, more or less, excluded from the membrane lipids of the seeds.
  • the present invention relates to genetically engineering of oil seeds, oleogeneous yeast and moulds to accommodate high amounts of uncommon fatty acids in their triacylglycerols by introducing genes coding for phospholipid hydrolases, below also called phospholipases, that specifically removes these fatty acids from the membrane lipids of the cell.
  • the inventors have identified phospholipase (phospholipase A2) enzymes respon ⁇ sible for the removal of medium chain fatty acids from phospholipids in plants.
  • the present invention relates to cDNA or genomic DNA coding for a phospholipid acyl hydrolase comprising a nucleotide sequence coding for an amino acid sequence with homology to Ulmus glabra phospholipase A2 as presented in Fig. 7 or amino acid sequences homologous to those encoded by the rice cDNA clones D49050, D47724, D47653 as presented in Fig. 6 and 7.
  • the invention relates to the use of a plant phospholipid hydrolase gene (cDNA or genomic DNA coding for a phospholipid hydrolase) in combination with a gene for an uncommon fatty acid for obtaining transgenic plants comprising both said genes.
  • a plant phospholipid hydrolase gene cDNA or genomic DNA coding for a phospholipid hydrolase
  • the enzyme encoded by said phospholipid acyl hydrolase gene, or cDNA is coding for a low molecular weight phospholipase A2 with distinct acyl specificity for uncommon fatty acids, such as medium chain, long chain (>C ⁇ 8 ), hydroxy, epoxy and acetylenic acids.
  • the invention relates to transgenic oil accumulating organisms comprising, in their genome, a plant phospholipid hydrolase gene having specificity for a particular uncommon fatty acid and the gene for said uncommon fatty acid.
  • said organisms are selected from the group consisting of oil crops, yeasts, and moulds.
  • the invention also relates to oils from such organisms.
  • Elm (Ulmus glabra) seed triacylglycerols are mainly composed of octanoic (8:0) and decanoic (10:0) acids, but these acids are very low in concentrations in the phospholipids of the seeds (Stahl et al., 1995).
  • Membrane fractions (microsomal preparations) from developing Ulmus glabra seeds had high phospholipase A2 (PLA2) activity towards phosphatidylcholine with medium chain fatty acids in position sn-2 (octanoic, decanoic and dodecanoic (12:0) acids but very low activity towards phosphatidylcholine with octadeca-9-enoic acid (oleic acid - a common fatty acids) (Stymne, 1993, Stahl et al. 1995). Microsomal preparations from developing rape seed did not have such phospholipase A2 activity towards medium chain fatty acids (Stahl et al. 1995).
  • phospholipase A2 If a gene coding for plant phospholipase A2 with specificities for a particular uncommon fatty acids is expressed in transgenic oil producing organisms engineered to produce that uncommon fatty acids, the recombinant phospholipase A2 will remove the uncommon fatty acids from the phospholipids of the cell and thereby prevent deleterious effects on cell metabolism caused by the presence of this acid in the membrane lipids.
  • This invention describes how such phospholipase A2 genes will be isolated and what uses they will have in commercial applications.
  • Fig.1 shows a SDS-polyacrylamid electrophoresis of soluble developing elm seed PLA2 purification fractions, followed by colloidal Coomassie staining.
  • Lane A and I contain 100 ng of MW standards (Pharmacia low MW);
  • lane B 100 OOOg supernatant, 50 ⁇ g;
  • lane C ammonium sulphate pellet, 50 ⁇ g;
  • lane D acetone supernatant, 50 ⁇ g;
  • lane E Q Sepharose, 40 ⁇ g;
  • lane F Superose 12, 25 ⁇ g; lane G, C4-HPLC, 2 ⁇ g; lane H C2C18-SMART, 100 ng;
  • lane J commercial Naja naja kouthia PLA2, 100 ng.
  • Fig.2 shows PLA2 activity measurements of gel pieces from whole lanes (5-94 KD) of a SDS-PAGE 8-18% gradient gel. Lane A contains 50 ng of Naja naja kouthia PLA2 (Sigma) and lane B 50 ng of developing elm seed soluble PLA2. PLA2 activity recovered from gel pieces of similar lanes are shown on each side.
  • Fig. 3 shows molecular weight data of the purified soluble PLA2, from MS-Malditof.
  • Fig. 4 shows molecular weight data of the purified soluble PLA2 that has been reduced and iodoacetamide alkylated, from MS-Malditof.
  • Fig. 5 shows a SDS-polyacrylamid electrophoresis of purified microsomal peak II PLA2 from developing elm seeds, with recovered PLA2 activity.
  • Lane A contain about 20 ng of purified peak II PLA2, non reduced;
  • lane B contain 25 ng of MW standard (Pharmacia low MW). The gel was silver stained.
  • Fig. 7 Alignment of the N-terminal sequence of the purified soluble PLA2 from elm seeds with deduced amino acid sequences from three EST-clones from rice green shoots, including the cDNA clone D49050 fully sequenced by the inventors.
  • the EST-sequences are denoted by their GenBank accession number. conserveed amino acid positions betweeen the elm and rice proteins as well as the regions with homology to the Ca2 + - binding and the active site in animal low molecular weight PLA2 ' s are boxed
  • a fourth rice clone (GenBank ID D47320) with high homology to the three above was found in the EST database, but excluded from the alignment due to lower quality of the DNA sequence
  • Membrane associated PLA2 activity was assayed according to Stahl et al (1995) using s/7-1 -palm ⁇ toyl-s7-2-[ ' ' 4 C]decanoyl-SA7-glycerol-3-phosphochol ⁇ ne as substrate
  • 1-palm ⁇ toyl-2-[ 14 C]palm ⁇ toyl-glycerol-s/?-3-phosphochol ⁇ ne was used as substrate and was presented as mixed micelles with the non-ionic detergent lubrol PX, in a PC/detergent molar relation of 1 10
  • Samples, 0 5-10 ⁇ l, were assayed for PLA2 activity by incubation at 30°C for 5-30 mm with 5 nmol of 1 Re ⁇ labelled phosphatidylcholine (10 000 dpm/nmol) in a total volume of 50 ⁇ l of 50 mM T ⁇ s/HCI, pH 8 0 containing 10
  • Protein fractions were if necessary concentrated in Sped-Vac and precipitated with ethanol/chloroform according to Wessel and Fl ⁇ gge (1984).
  • Precipitated proteins were pelleted by centrifugation 10 000 g for 10 min and resuspended in 130 ml of 50 mM dietanolamin buffer pH 8.5. Ice cold acetone was added to a final concentration of 45 % (v/v) and the extract was left at 4°C for 30 min. Precipitated proteins were removed by centrifugation for 10 min at 10 000 g and the resulting supernatant was dialysed against 20 volumes of 20 mM piperidin, pH 11.0, with one change over night. The dialysed extract was applied to a Q-Sepharose Fast Flow 7 ml column (1.0 x 10.0 cm) equilibrated in 20 mM piperidin, pH 11.0.
  • the column was eluted with a linear salt gradient from 100 to 500 mM NaCl in 20 mM piperidin, pH 11.0 at a flow rate of 2 ml/min. 3 ml fractions were collected and assayed for PLA2 activity. A single broad peak of activity was eluted at a salt concentration of 200 to 300 mM NaCl. Peak fractions were pooled, concentrated on Centricon-10 to 0.6 ml and chromatographed in three separate runs on a Pharmacia Superose 12 (1.0 x 30.0 cm) gel filtration column (0.4 ml/min) in 20 mM Tris/HCI, 150 mM NaCl, pH 8.0.
  • the PLA2 was finally purified to apparent homogeneity on a C2C18 reversed-phase HPLC column (0.21 x 10.0 cm) equilibrated in 0.1 % TFA and developed at 100 ⁇ l/min with a 60 min gradient (30-60% acetonitrile in 0.1 % TFA) using a SMART system (Pharmacia). Peaks monitored at 280 nm were automatically collected and then subjected to PLA2 assay. The PLA2 elutes as a discrete peak in the gradient at about 50% acetonitrile.
  • the PLA2 was purified about 180 000 times from the developing elm seed extract of soluble proteins, to a final specific activity of 44 ⁇ mol/min x mg protein (see table I).
  • the tblastn search program against Non-redundant GenBank+EMBL+DDBJ+ PDB sequences and Non-redundant Database of GenBank EST Division the best aligned sequences are three EST's (GenBank accession number D47724, D47653 and D47320) derived from green rice shoots.
  • Fig. 7 shows an alignment of the amino-terminal sequence with the deduced amino-acid sequence from two of these EST-clones and the D49050 rice EST- clone.
  • the amino-terminal sequence show significant homology with the rice sequences, notably the positions of the three cysteine-residues are conserved.
  • predictions of leader peptide cleavage site of rice clones D47724 and D47653 suggest cleavage between G 24 and L 25. This supports the alignment of the amino-terminal sequence to the mature part of the rice sequences. Regions with high homology to the conserved Ca 2+ binding- and active sites of secretory PLA2's (see fig 6) are both found in all three alined rice EST's.
  • 60 g of liquid nitrogen frozen elm endosperm was homogenized with a ultraturrax® in 600 ml of ice cold 100 mM potassium phosphate buffer, pH 7.2 containing 0.33 M sucrose.
  • the homogenate was filtered through two layers of Miracloth® and centrifugated 10 000 g for 12 min.
  • the supernatant was filtered through one layer of Miracloth® and centrifugated a second time, at 100 000 g for 90 min.
  • the microsomal pellets were resuspended in 90 ml of 100 mM potasium phosphate, pH 7.2 with a glass homogenizer and, if not used immediately, stored at -80° C.
  • microsomal membranes were diluted to 150 ml with 100 mM potasium phosphate, pH 7.2 and solubilized by the addition of 150 ml of 200 mM potasium phosphate, pH 7.2 containing glycerol 17% (v/v), lubrol PX 0.6% (w/v) and EGTA 1 mM.
  • the mixture was incubated 15 min at 4° C followed by a centrifugation 100 000 g at 4° C for 90 min.
  • the supernatant was dialysed against two changes of 5 liter of 20mM dietanolamin, pH 8.5 containing glycerol 8.7% (v/v) and lubrol PX 0.06% (w/v).
  • the dialysed supernatant was passed through a 200 ml Q-Sepharose column (50 x 100 mm) at a flow rate of 3 ml/min.
  • Non-retained material with about 30-50 % of the PLA2 activity was collected and pH adjusted to 5.7 by adding requiring amount of 1 M MES buffer.
  • This fraction was applied to a 7 ml SP-Sepharose column (10x 100 mm) equilibrated in 50 mM MES, pH 5.7 with glycerol 8.7% (v/v) and lubrol PX 0.06%(w/v) at a flow rate of 3 ml/min.
  • the column was washed with several column volumes of equilibrating buffer and then eluted with a 100 ml linear gradient from 0 to 480 mM of NaCl in the same buffer. Eluated fractions containing PLA2 activity were pooled and concentrated to a volume of 200 ⁇ l by centrifugation on Centricon-50 and vacuum evaporation in a Speed-Vac concentrator (Savant). The sample was applied to a Superose 12 (10 x 300 mm) Pharmacia column equilibrated in 20 mM Tris, pH 8.0 with glycerol 4.3% (v/v), lubrol PX 0.06% (w/v) and 50 mM NaCl.
  • Fractions with PLA2 activity were pooled and further purified using a C4 reversed-phase HPLC column (Vydac 0.46 x 10.0 cm) that was equilibrated in 0.1 % trifluoroacetic acid (TFA).
  • the column was developed at 0.4 ml/min with a 30 min gradient (20-45% of acetonitrile in 0.1 % TFA) and peaks monitored at 280 nm were collected manually.
  • microsomal PLA2 activity was purified from the microsomal fraction with a specific activity of 0 28 nmol/min x mg protein to a specific activity of about 50 ⁇ mol/min x mg protein which gives a purification factor of about 100 000
  • the purified soluble and microsomal PLA2S have very similar properties They have a pH optimum between 7 and 9, an absolute requirement for Ca2+ for activity with several mM for optimal activity
  • the activities are extremly stable both to extreme pH values, heat and organic solvents
  • the activities are, however, sensitive to reducing agents like DTT and mercaptoethanol (see Table II)
  • EGTA I O mM 170 The purified PLA2's hydrolyses the sn-2 position of phospholipids (Table III), and does not show any activity towards diacylglycerols or lysophosphatidylcholine (Table IV).
  • PC phosphatidylcholine
  • LPC sn-1- lysophosphatidylcholine
  • DAG Diacylglycerol
  • the molecular weight and the biochemical characteristics of both the soluble and microsomal elm PLA2 suggest that they are related to the well described low MW "secretory" PLA2S from animal sources. This is further supported by the amino- terminal sequence data and alignments.
  • the secretory PLA2's have all conserved amino acid sequences at the Ca 2+ bindning site and at the active site as well as cyein residues.
  • searching databases for deposited expressed sequences from plants with homology to low molecular weight animal phospholipases in Ca 2+ binding site and active site the inventors found three anonymous partially sequenced cDNA clones from green shots of (GenBank ID: D49050, D47724, D47653).
  • the cDNA clone D49050 was received upon request from Dr. Yoshiaka Nagamura, DNA Materials Management group, Rice Genome Project, NIAR/STAFF, STAFF Institute, 446-1 , Ippaizuka, Kamiyokoba Tsukuba, Ibaraki 305 Japan.
  • the entire cDNA was sequenced and was shown to contain an open reading frame encoding a full length protein of an estimated molecular weight of 15 kDa.
  • An alignment of the deduced amino acid squence of D49050 with a number of animal low molecular weight PLA2s is presented in Fig. 6.
  • the D49050, D47724, D47653 clones coded for proteins with the same amino acid sequences as in thee Ca 2 + binding site and active site in the animal low molecular weight PLA2s and similar to these enzymes they contained several cystein residues (see Fig. 7).
  • the cDNA clones also coded for amino acid sequences with significant homologies with the N-terminal sequence of the purified phospholipasee A2 from elm seeds where the positions of the three cystein residues of the elm enzyme was totally conserved in all three cDNAs (see Fig.7). Thus with all probability these rice cDNAs were coding for a plant PLA2 similar to the enzyme purified from developing elm seeds according to the invention.
  • a recombinant PLA2 protein By expressing this cDNA in suitable organism, like bacteria, for example E.coli, yeast or plants, a recombinant PLA2 protein will be obtained and PLA2 activities can be demonstrated.
  • suitable organism like bacteria, for example E.coli, yeast or plants
  • PLA2 activities can be demonstrated.
  • suitable organism like bacteria, for example E.coli, yeast or plants
  • PLA2 activities can be demonstrated.
  • the physiological function of the rice enzyme is unknown, a function in rice shoots could be removal of oxygenated fatty acids from membrane lipids, as has been shown to take place in e.g. wheat roots (Banas et al, 1992).
  • degenerated nucleotide primers based on suitable amino acid sequences of the soluble elm PLA2 and rice cDNA clones amplification of elm fragments containing the corresponding sequences will be done from cDNA or genomic DNA from elm seeds by PCR. These fragments will be used as probes for screening for the elm cDNA PLA2 from a cDNA library from developing elm seeds.
  • homologous cDNA coding for PLA2 with other acyl specificities than the elm enzyme can be isolated from other plant species with the aid of the cDNA encoding for the elm PLA2 and/or the rice cDNA clones as probes Alternatively suitable amino acid sequences of these enzymes can be used to construct degenerated nucleotide primers and amplify cDNA fragments derived from the other plant species. These fragments will be used as probes for screening for the cDNA coding for PLA2 from a cDNA library from other plant species.
  • this cDNA can be used for transformation.
  • the PLA2 gene i.e. the PLA2 cDNA or genomic clone
  • a gene for an uncommon fatty acid for obtaining transgenic plants comprising both said genes.
  • the transgenic plants are obtained by using said plant phospholipid acyl hydrolase gene for transforming transgenic oil accumulating organisms engineered to produce said uncommon fatty acid.
  • transgenic plants are obtained by using the plant phospholipid acyl hydrolase gene for transforming oil accumulating organisms, which are crossed with other oil accumulating organisms engineered to produce said uncommon fatty acid.
  • Plant microsomal phospholipases exhibit preference for phosphatidylcholine with oxygenated acyl groups. Plant Science 84, 137-144
  • Plant microsomal phospholipid acyl hydrolases have selectivities for uncommon fatty acids. Plant Physiol. 107, 953-962

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  • Molecular Biology (AREA)
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Abstract

Cette invention concerne l'utilisation d'un gène d'hydrolase phospholipidique de plante en combinaison avec un gène, un acide gras non commun en vue d'obtenir des plantes transgéniques comprenant ces deux gènes. Cette invention concerne également des organismes accumulant de l'huile transgénique qui ont, dans leur génome, un gène d'hydrolase phospholipidique de plante présentant une spécificité pour un acide gras non commun spécifique et le gène pour ledit acide gras non commun, ainsi que des huiles de graines provenant de ces organismes.
PCT/SE1997/000554 1996-03-29 1997-03-27 Enzyme de plante et utilisation de cette derniere WO1997037006A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU25256/97A AU2525697A (en) 1996-03-29 1997-03-27 Plant enzyme and use thereof
EP97916701A EP0904358A1 (fr) 1996-03-29 1997-03-27 Enzyme de plante et utilisation de cette derniere

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9601237-2 1996-03-29
SE9601237A SE9601237D0 (sv) 1996-03-29 1996-03-29 Use of a plant enzyme gene for transformation

Related Child Applications (2)

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US09155124 A-371-Of-International 1999-03-02
US09/917,805 Continuation US20040073973A1 (en) 1996-03-29 2001-07-31 Plant enzyme and use thereof

Publications (1)

Publication Number Publication Date
WO1997037006A1 true WO1997037006A1 (fr) 1997-10-09

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EP (1) EP0904358A1 (fr)
AU (1) AU2525697A (fr)
CA (1) CA2250233A1 (fr)
SE (1) SE9601237D0 (fr)
WO (1) WO1997037006A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040527A1 (fr) * 2000-09-18 2002-05-23 Zhongshan University Nouvelle lapemis hardwicki phosphatidase a2 et gene codant ce polypeptide
WO2006008099A3 (fr) * 2004-07-16 2007-03-15 Basf Plant Science Gmbh Procede permettant d'augmenter la teneur d'organismes transgeniques en acides gras a chaine longue polyinsatures

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001565A1 (fr) * 1992-07-01 1994-01-20 The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Genes permettant de modifier le metabolisme des plantes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001565A1 (fr) * 1992-07-01 1994-01-20 The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Genes permettant de modifier le metabolisme des plantes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIPIDS, Volume 31, No. 8, August 1996, MICHAEL G. KOCSIS et al., "Phosphatidate Phosphatases of Mammals, Yeast and Higher Plants", pages 785-802. *
PLANT PHYSIOL., Volume 107, 1995, ULF STAHL et al., "Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids", pages 953-962. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040527A1 (fr) * 2000-09-18 2002-05-23 Zhongshan University Nouvelle lapemis hardwicki phosphatidase a2 et gene codant ce polypeptide
WO2006008099A3 (fr) * 2004-07-16 2007-03-15 Basf Plant Science Gmbh Procede permettant d'augmenter la teneur d'organismes transgeniques en acides gras a chaine longue polyinsatures
US7842852B2 (en) 2004-07-16 2010-11-30 Basf Plant Science Gmbh Method for increasing the content of polyunsaturated long-chained fatty acids in transgenic organisms

Also Published As

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SE9601237D0 (sv) 1996-03-29
AU2525697A (en) 1997-10-22
EP0904358A1 (fr) 1999-03-31
CA2250233A1 (fr) 1997-10-09

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