AU4756500A - Gene isolated from (ricinus communis) encoding a new protein that interacts withthe oleate 12-hydroxylase enzyme - Google Patents

Description

WO 00/70052 PCT/EPOO/04181 GENE ISOLATED FROM RICINUS COMMUNIS ENCODING A NEW PRO TEIN THAT INTERACTS WITH THE OLEATE 12-HYDROXYLASE EN ZYME. The present invention relates to the identifica 5 tion and characterization of a gene of Ricinus communis (R.communis) which encodes for a protein capable of in teracting with the oleate 12-hydroxylase enzyme that catalyzes the introduction of a hydroxyl group into the molecule of oleic acid transforming it into ricinoleic 10 acid. The invention also relates to means and methods for producing transgenic plants with a modified compo sition of fatty acids. Ricinoleic (12-hydroxy-9-octadecenoic) acid is a 15 monohydroxylated fatty acid whose only commercial source is seed oil synthesized in the endosperm of ripe seeds of R.communis, where it represents about 90% of hydroxylated fatty acids. Studies in vivo with radioactive tracers indicate 20 that, in the endosperm of unripe seeds of R.communis, ricinoleic acid (also known with the term ricinoleate) is synthesized by the direct substitution of a double bond of oleic acid with a hydroxyl group (Morris, L.J. 1967, Biochem. Biophys. Res. Commun. 29, 311-315) . This WO 00/70052 PCT/EP0O/04181 2 reaction is catalyzed by the oleate 12-hydroxylase en zyme whose activity seems to be associated with the en doplasmic reticulum. Enzymatic tests indicate that the substrate for 5 oleate 12-hydroxylase is the oleic acid esterified with lecithin or with another phospholipid; the esterified ricinoleate is released from the lipid complex owing to the intervention of a phospholipase A, specific for fatty acids oxygenated in the presence of molecular 10 oxygen, NAD(P)H and cytochrome b5. NAD(P)H is required for reducing the cytochrome b5, intermediate electron donor for the hydroxylase reaction (Bafor M. Et al., (1991), Biochem J., 280, 507-514; Smith M.A. et al., (1992), Biochem J, 287, 141-144). The hydroxylated 15 fatty acid is then transferred, by means of the Kennedy pathway, to the pathway of triacylglycerols where it accumulates. Ricinoleic acid, owing to the presence of the hy droxyl group, is one of the most versatile natural 20 products and has numerous industrial and food applica tions. In particular, ricinoleic acid can be used in the production of paints, polymers such as nylon-11, drugs, lubricants, cosmetics, resins and other materi als.

WO 00/70052 PCT/EPOO/04181 3 The production of ricinoleic acid however is lim ited by the high susceptibility to climatic variations of the cultivations of R.communis plants and by the toxicity of ricin, an allergen present in castor beans. 5 The possibility of producing ricinoleic acid in vegetable species which are more tolerant towards cli matic variations and which do not contain toxic sub stances would allow a larger and simpler production and application of the acid itself. 10 For this purpose, the gene that encodes the oleate 12-hydroxylase enzyme has recently been isolated and used to transform plants such as Nicotiana tabacum, Arabidopsis thaliana, Linum usitatissimum and Brassica napus. 15 In all these cases, however, although a modified content of fatty acids was observed, the increase in ricinoleic acid was low if not zero (Broun P. and Somerville C., 1997, Plant Physiol, 113, 933-942). It is known that in many biological processes, 20 such as replication, transcription or metabolism, enzy matic complexes, whose action is correlated to the co operation of various proteic subunits, intervene. For example, evidence of the possible interaction of various proteins in desaturation processes was ob- WO 00/70052 PCT/EPOO/04181 4 tained from studies on the activity of the toluene 2 mono-oxygenase enzyme isolated from the bacterium Burk holderia cepacia (Newman L.M. et al., 1995, Biochemis try, 34, 14066-14076). 5 It was therefore assumed that the hydroxylase of vegetable fatty acids can also form part of a multicom ponent system and that the hydroxylase activity of the oleate 12-hydroxylase enzyme of R.communis consequently requires the intervention of further co-factors or pro 10 teins. A gene of R.communis which encodes for a new pro tein capable of interacting with the oleate 12 hydroxylase enzyme, has now been identified and charac terized. This gene can be used in genetic transforma 15 tion programs of plants containing the oleate 12 hydroxylase enzyme to favour the production of ricino leic acid. In accordance with this, an objective of the pres ent invention is the cloned and sequenced gene which 20 encodes a protein capable of interacting with oleate 12-hydroxylase. A second objective of the present invention is an expression recombinant vector in host cells comprising said gene.

WO 00/70052 PCT/EPO0/04181 5 A further objective of the present invention is a host microorganism transformed with said vector. Yet another objective of the present invention re lates to transgenic plants transformed with said vec 5 tor. Additional objectives of the present invention will appear evident upon reading the description and examples. Brief description of the figures 10 Figure 1: Southern Blot of the genomic DNA of different species digested with the restriction enzyme EcoRI and hybridized with the 762 bp fragment of the plasmid pTargl marked with 32p. The hybridization signal corre sponds to a gene in a single copy only evident in 15 R.communis. Figure 2: Northern blot of messenger RNA extracted at various development stages of the seed (10, 20, 30, 35 and 40 DAP), from the leaves, stem and roots of the R.communis plant. The filter was hybridized with the 20 762 bp fragment of the plasmid pTargl marked with 3P. The presence of an mRNA with a molecular weight of about 1.0 Kb, is observed in unripe seeds at 10 DAP and 20 DAP and a transcript with larger dimensions in the leaves.

WO 00/70052 PCT/EPOO/04181 Figure 3: Northern blot of messenger RNA extracted at different development stages of the seed (10, 20, 30, 35 and 40 DAP), from the leaves, stem and roots of the R.communis plant. The filter was hybridized with the 5 1216 bp fragment of oleate 12-hydroxylase marked with 32P. The hybridization signal, of about 1.6 Kb, is pres ent in the unripe seeds at 20, 30, 35 and 40 DAP. Detailed description of the invention The isolation of nucleotide sequences which encode 10 for proteins of interest can be carried out by known techniques. In particular, to isolate new proteins that inter act with oleate 12-hydroxylase in unripe seeds of R.communis, the "HybridZap two hybrid vector" of 15 Stratagene was used, a eukaryotic system (Saccharomy ces cerevisiae) which enables new genes to be identi fied in vivo, that encode proteins which interact with a known protein (Fields S. et al., 1989, Nature, 340, 245-246) . This system exploits the characteristics of 20 the transcriptional activator GAL4 of S.cerevisiae, which regulates the expression of genes that encode en zymes involved in the galactose metabolism. GAL4 consists of two domains separable and func tionally essential for its activity; an N-terminal do- WO 00/70052 PCT/EPOO/04181 7 main (Binding Domain, BD), which is linked to specific sequences of the DNA (UAS: upstream activating se quences), and a C-terminal domain, containing acid re gions (Activation Domain, AD), which is necessary for 5 the transcriptional activation. The system used allows two hybrid proteins to be generated, containing the functional domains of GAL4, i.e. the Binding Domain fused with a known protein which acts as bait, and the Activation Domain fused 10 with unknown proteins (target) from an expression li brary. If the known protein interacts with a target pro tein forming a protein-protein complex, the two func tional domains of GAL4 are brought under optimum condi 15 tions and activate the transcription of the reporter gene lac-Z, whose product is shown by means of colori metric reaction. The total RNA was extracted from a pool of unripe seeds of R.communis using the "Hot-Phenol" method and 20 the polyadenilate messenger RNA (mRNA) was isolated with oligo-dT columns (Pharmacia) . The cDNA encoding the oleate 12-hydroxylase enzyme was subsequently pre pared by applying the polymerase chain reaction (PCR) technique on the mRNA using, as primers, a pair of oli- WO 00/70052 PCT/EPOO/04181 8 gonucleotides having the sequences that flank the en coding region comprising the start and stop translation codon of said gene. As the interaction of target proteins can take 5 place with the whole protein under examination or with parts of it, it was decided to clone both the whole gene encoding for oleate 12-hydroxylase and its termi nal regions 5' and 3', in the plasmid pBD-GAL4, which contains the sequence that encodes for the Binding Do 10 main. The three DNA inserts were first amplified with the appropriate primers having restriction sites EcoRI for the Forward primer and Sall for the Reverse primer, digested with the above enzymes, directionally inserted 15 into the vector pBD-GALA4 predigested with the same en zymes and introduced into the competent cells Epicurian coli XL1-Blue. The recombinant clones, containing the expected fragments, were characterized by means of restriction 20 analysis and their identity was confirmed with the se quence reactions carried out using the Taq Dye Deoxy Terminator Cycle Sequencing kit (Perkin Elmer) and ana lyzed with the automatic sequencer. A cDNA library was then prepared from unripe seeds of R.communis in the X WO 00/70052 PCT/EPOO/04181 9 HybridZap phage vector which expresses consistent hy brid proteins of the activation domain of GALA4 and proteins of R.communis. In practice, the polyadenilate mRNA of R.communis 5 was used for the synthesis of double filament cDNA op erating according to the protocols suggested by the Kit distributor (Stratagene). The molecules of cDNA having a high molecular weight, useful for the construction of the library, 10 were then separated from those having a low molecular weight, which represent the fraction of molecules in which the synthesis is incomplete. The fraction of high molecular weight cDNA was in serted into the H HybridZap phage vector and packed 15 with the packaging extracts containing proteins for the head and tail of the phage. The dimensions of the in serts present in the library produced were checked by means of PCR and amplified with a specific pair of primers for the vector pAD-GAL4. The results obtained 20 demonstrated that the fragments of the cDNA library had an average dimension of 1.4 Kb. After amplification, the library in the lambda phage was converted to a plasmid library by excision in vivo.

WO 00/70052 PCT/EPOO/04181 10 The library was subsequently multiplied in the strain of E.coli XLOR and the plasmid DNA was extracted and co-transformed, in separate co-transformation proc esses, with the DNA extracted from the bait plasmids 5 containing the whole gene and parts of oleate 12 hydroxylase, using yeast cells (YRG-2 strain), having reporter genes his3 and lacZ. From the test of the three sequences of oleate 12 hydroxylase with the expression library, colonies of 10 yeast were identified which had the typical blue colour of the lacZ gene activity, thus demonstrating the prob able interaction of the bait protein with an unknown target protein. Subsequent analyses on these colonies enabled a 15 "positive" co-transformed yeast clone to be identified, which activated the transcription of both reporter genes. This indicated the complete interaction between the N-terminal region of oleate 12-hydroxylase and an unknown Target protein. 20 The plasmid isolated from this positive yeast clone was indicated with the abbreviation pTargl. The interaction specificity between oleate 12 hydroxylase and the new protein identified, was con firmed by co-transformation experiments in S.cerevisiae WO 00/70052 PCT/EPOO/04181 11 YRG-2 yeast cells with bait plasmids containing the whole oleate 12-hydroxylase gene and the 5' terminal portion of this gene, respectively. Two yeast colonies were identified from tests of the two sequences, one 5 for each co-transformation process, which had the typi cal blue colour of the lacZ gene activity. To confirm the presence and dimensions of the bait proteins and to verify the presence and dimensions of the gene encoding the protein Targl2 identified, PCR 10 analyses were carried out on the DNA extracted from the yeast clones resulting positive from the expression test of the reporter gene lacZ, using specific primers for the Binding Domain region and for the Activation Domain region. 15 The authenticity of the amplification products ob tained was demonstrated not only by the size of the ex pected fragments, but also by the hybridization analy sis carried out on the latter. In addition, the plasmid DNA of pTargl was iso 20 lated from the yeast colony and the cDNA insert was pu rified with the "Double GeneClean" kit (BIO 101 Inc., U.S.A.). The purified fragment was subsequently cloned in the vector pGEM-T (Promega) and then introduced into competent cells of E.coli DH5a.

WO 00/70052 PCT/EP0O/04181 12 The plasmid DNA isolated from the recombinant clones was subjected to sequence analysis with a Taq Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer), using the automatic sequencer ABI 373A (Perkin 5 Elmer). From the sequence analyses carried out on the DNA of the insert of the plasmid pTargl, it can be observed that the fragment isolated has a dimension of 762 bp and contains an Open Reading Frame (ORF) of 540 bp pre 10 ceded by 75 bp at 5' and followed by 147 bp at 3'. The ORF encodes a protein of 180 aminoacids with a molecu lar weight of 19.8 KDa. The nucleotide and aminoacid sequences were com pared with the sequences available on data banks by 15 means of FASTA and BLAST analyses, and homologous se quences were not found, indicating the uniqueness of the DNA tract of R.communis and the uniqueness of the protein identified. To verify the identity of the protein capable of 20 interacting with oleate 12-hydroxylase, analyses were carried out on the genomic DNA of different species. The results showed the presence of a signal only in R.communis. This demonstrates that the gene isolated is specific of the genome of R.communis and is not an out- WO 00/70052 13 PCT/EPOO/04181 come of the system adopted for its identification. In addition, expression analyses were carried out on the messenger RNA extracted at different development stages of the seed (10, 20, 30, 35 and 40 days after 5 pollination), from the leaves, stem and roots of the R.communis plant. The results of the hybridization of the Northern blot with the fragment of pTargl marked with 32 P showed the gene expression in the leaves and unripe seeds 10 10 and 20 days after pollination. The same filter was hybridized with the fragment of oleate 12-hydroxylase which begins to be expressed in the unripe seeds at 20 DAP, where the signal is very weak, subsequently increasing its expression in the 15 stages at 30, 35 and 40 DAP. As expected, there was no hybridization signal in the samples of RNA correspond ing to leaves, stem and roots. On the basis of these results, it can be concluded that the new protein most probably intervenes in the 20 first development stages of the R.communis seed at the beginning of the synthesis of ricinoleic acid, carrying out a regulating action. The gene of the present inven tion can be cloned in an expression vector in plants, by putting it under the control of suitable regulation WO 00/70052 PCT/EP0O/04181 14 sequences (promoter and terminator). Vectors suitable for the purposes of the present invention are for example those deriving from the Ti plasmid of Agrobacterium tumefaciens as described by 5 Bevan M., (1984), Nucleic Acid Research 12: 8711-8721. These vectors are used to transform the plants by means of conventional methods. The method described by G. An et al. (Binary vectors, Plant Molecular Biology Manual A3, Kluwer Academic, Dordrecht, pages 1-19, 10 1988), which is based on the capacity of Agrobacterium tumefaciens to transfer part of its own DNA to vegeta ble cells, is preferably used. The plasmid pTargl containing the gene of the pre sent invention was deposited as E.coli DH5aX/MA292 at 15 the CentraalBureau Voor Schimmelcultures where it re ceived the deposit number CBS 101642. The following examples, whose sole purpose is to provide a more detailed description of the present in vention, should in no way be considered as limiting the 20 scope of the invention itself. Example 1 RNA Isolation The total RNA was extracted from a pool of unripe seeds (10-40 days after pollination) of Ricinus commu- WO 00/70052 15 PCT/EPOO/04181 nis by means of the "Hot-Phenol" method described by Shirzadegan M. et al. (1991), Nucl. Acids Res.: 19, 6055, to which several modifications were made. In short, 2 g of vegetable material were crushed 5 in liquid nitrogen and then suspended in 6 ml of ex traction buffer (0.1 M LiCl, 0.1 M Tris-HCl pH 7.6, 0.01 M EDTA, 1% Sodium dodecylsulfate (SDS), phenol) preheated to 800C. The suspension was incubated at 80*C for 5 minutes and 3 ml of a mixture of chloro 10 form/isoamyl (24:1, v/v) were added. The sample was vortex mixed and subsequently centrifuged at 12,000 rpm, at 40C for 15 minutes. The aqueous phase was re covered, subjected to an additional extraction cycle with phenol/chloroform/isoamyl (25:24:1) and centri 15 fuged under the same conditions specified above. The supernatant was recovered and the RNA precipitated by the addition of a volume of LiCl 4 M. The sample was incubated at -20'C for a night and then centrifuged at 13,000 rpm, at 4 0 C for 30 minutes. 20 The RNA pellet was re-suspended in water, trans ferred to microcentrifuge tubes and precipitated by the addition of LiCl 4 M and 0.2 volumes of EDTA 0.5 M. Af ter centrifugation at 13,000 rpm, at 40C for 30 min utes, the pellet was suspended again in water and pre- WO 00/70052 PCT/EPOO/04181 16 cipitated with 0.1 volumes of NaCl 5 M and 2.5 volumes of ethanol 100%. After centrifugation at 15,000 rpm, at 4*C for 30 minutes, the pellet was recovered, washed twice with ethanol 70%, dried and re-suspended in wa 5 ter. Example 2 Isolation of the cDNA encoding for oleate 12 hydroxylase The polyadenilate messenger RNA (poliA-RNA) was 10 prepared from the total RNA obtained in example 1, us ing oligo-dT columns (Pharmacia) according to the in structions of the distributor. 3.5 ptg of polyadenilate messenger RNA were then used for the synthesis of double filament cDNA using 15 the Kit distributed by PHARMACIA, operating under the experimental conditions suggested by the supplier of the kit. On the basis of the sequence of oleate 12 hydroxylase deposited in the data bank (GenBank, AC 20 U22378) the following oligonucleotides were synthe sized: (1) 5'GGA TCC CTC AGG AAA GTG CTT A 3' (FORWARD) (2) 5' TCT AGA CAT TCC TTC TTG TTC TAA TT3' (REVERSE) These oligonucleotides, which correspond to the WO 00/70052 PCT/EPOO/04181 17 regions that flank the portion encoding the enzyme com prising the start and stop translation codon, were used as primers for the isolation of the fragment corre sponding to oleate 12-hydroxylase by means of the poly 5 merase chain reaction (PCR) technique. The amplification was effected in a DNA Thermal Cycler 480 apparatus (Perkin Elmer Cetus) using a reac tion mixture (25 pl) containing 6 pl of double filament cDNA, 10 mM Tris HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 10 2.5 FLM of each primer, 0.1 mM of dNTP and 2.5 polymer ase Taq Units (Boheringer). After a first denaturation cycle for 5 minutes at 95'C, the reaction was continued with the following cy cles: 15 1 minute at 94'C (denaturation) 1 minute at 56'C (pairing) 2 minutes at 72 0 C (lengthening) for a total of 35 cycles, followed by 10 minutes at 72'C (final extension). 20 The amplification product, corresponding to a fragment of about 1200 base couples, was separated on agarose gel at 1.0%, the DNA band of interest was re covered and purified with the GeneClean kit (BIO 101 Inc, U.S.A.) . About 100 ng of the DNA thus isolated WO 00/70052 PCT/EPOO/04181 18 were ligated to 50 ng of pGEM-T plasmid (Promega) in 10 pl of reaction mixture, in the presence of 2 units of T4 DNA ligase, at 40C for a night. 5 pl of this mixture were used to transform compe 5 tent cells of E.coli DH5a (BRL). The transforming agents were selected on plates of LB medium (NaCl 10 g/l, Yeast extract 5 g/l, Bacto-triptone 10 g/l and agar 20 g/l) containing 50 pig/ml of ampicillin. The plasmid DNA extracted from 6 positive clones 10 was subjected to sequence analysis to verify the nu cleotidic correspondence with the gene of oleate 12 hydroxylase isolated by Van de Loo F. Et al., 1995, PNAS, 92, 6743-6747. The reactions and sequence analy ses were carried out with the Taq Dye Deoxy Terminator 15 Cycle Sequencing TM kit (AB-PEC) using an ABI Prism 373A DNA Sequencer (AB-PEC). One of the plasmids analyzed, containing a frag ment of DNA analogous to the published sequence SEQ:ID Nr. 1, was called pC18-MA. 20 Example 3 Construction of the "bait" vectors (pBD-GAL4) As the interaction of target proteins can take place with the whole protein under examination (bait) or parts of this, it was decided to clone both the WO 00/70052 19 PCT/EPOO/04181 fragment corresponding to the whole gene encoding for oleate 12-hydroxylase, and also the fragments corre sponding to the regions 5' and 3' of said gene, in the plasmid pBD-GAL4 (Stratagene). 5 For this purpose four oligonucleotides were syn thesized, of which the Forward primers (abbreviated as F) have the restriction site EcoRI and the Reverse primers (abbreviated as R) the site Sall. The nucleo tide sequences of the primers are as follows: 10 (a) 5'GAA TTC CGC ATG TCT ACT GTC 3' (Forward, HydGal F) (b) 5'GTC GAC CAT TCC TTC TTG TTC 3' (Reverse, HydGal R) (c) 5'GTC GAC GCG ATC GTA AGG 3' (GalHydi-R) and 15 (d) 5'GAA TTC AAT GTC TCT GGT AGA C 3' (GalHydi-F) The primers HydGal-F and HydGal-R were used to am plify the whole gene of oleate 12-hydroxylase. The primers HydGal-F/GalHydi-R and HydGal R/GalHydi-F were used to amplify the region 5' of 624 20 bp (SEQ: ID Nr:2) and the region 3' of 633 bp (SEQ: ID Nr:3) of the gene of oleate 12-hydroxylase, respec tively. The amplifications with the above primers were carried out on 20 ng of the fragment of oleate 12- WO 00/70052 PCT/EPOO/04181 20 hydroxylase previously cloned and sequenced. The amplification products having the expected di mensions were digested with 10 units of restriction en zymes EcoRI and SalI (Boheringher) , separated on aga 5 rose gel 1% and the fragments of DNA of interest were then recovered and purified with the GeneCleanTm kit (BIO 101 Inc.). About 100 ng of each fragment were ligated, sepa rately, with the plasmid pBD-GAL4 linearized with the 10 enzymes EcoRI and SalI, in 10 ml of reaction mixture, in the presence of 2 units of T4 DNA ligase, at 4 0 C for a night. The ligase mixtures were used to transform competent cells of Epicurian coli XL1 Blue (Strata gene) . The recombinant clones were selected on plates 15 of LB medium to which 30 pg/ml of chloramphenicol had been added. The following recombinant "bait" plasmids were identified, which contain a fragment which con sists of the Binding Domain of Gal4 condensed with: (a) the whole gene of oleate 12-hydroxylase (pBD 20 GALA4/C18); (b) the 5-terminal region of the gene of oleate 12 hydroxylase (pBD-GALA4/C18-5); and (c) the 3-terminal region of the gene of oleate 12 hydroxylase (pBD-GALA4/C18-3).

WO 00/70052 PCT/EPOO/04181 21 The plasmids were characterized by restriction analysis and their identity was confirmed by effecting sequence reactions with the Taq Dye Deoxy Terminator Cycle sequencing kit (Perkin Elmer) and analyzed with 5 the automatic sequencer ABI 373A (Perkin Elmer). Example 4 Construction of the target library For the construction of a "target" library made up of hybrid proteins consisting of the activation domain 10 of GALA4 and proteins of unripe seeds of R.communis, the HybridZap phage vector was used. The experimental conditions adopted were those suggested by the supplier of the kit (Stratagene, HybridZap m Two-Hybrid cDNA gapack cloning kit, catalogue Nr.: 235612). 15 About 5 ptg of polyadenilate mRNA of R.communis were used for the synthesis of the double filament cDNA. The ends of the cDNA molecules, to which 3.6 pg of a linker having the restriction site EcoRI had been 20 added, were then blunted by the action of the polymerase DNA Pfu (Stratagene), and subjected to digestion with the enzyme XhoI (120 units), whose site is present in the primer polydT used for the synthesis of the first cDNA WO 00/70052 PCT/EPOO/04181 22 filament. This gave rise to molecules having the EcoRI site at one end and the XhoI site at the other end. The sample of cDNA was then passed on a Sephacryl S-500 column equilibrated in 20 mM Tris-HCl pH 7.5, 10 5 mM EDTA, 100 mM NaCl, and subjected to centrifugation for 2 minutes at 400 revs. Three fractions were recovered, whose molecular weight was verified by means of electrophoresis on non denaturing polyacrylamide gel at 5% (Sambrook, J. Et. 10 Al., (1989), Cold Spring Harbor Laboratory Press). About 100 ng of the first fraction of cDNA, corre sponding to that with a high molecular weight, were li gated with 1 p.g of the k HybridZap phage vector predi gested with the enzymes EcoRI and XhoI and packed with 15 packaging extracts containing proteins for the head and tail of the phage. The total quantity of phage parti cles obtained by the packaging in vitro was determined by plating aliquots with the strain XL1-Blue MRF'. The primary library obtained contains a total of 20 1.4 x 106 plaque forming units (pfu) per pg of ligated vector arms and 97% of these contains the DNA insert. The dimensions of the library produced were verified by subjecting to PCR reaction, 20 phage plaques, selected at random, and amplified with the specific pair of WO 00/70052 PCT/EPOO/04181 23 primers for the vector pAD-GAL4: a) 5'AD primer: 5' AGG GAT GTT TAA TAC CAC TAC3' b) 3'AD primer: 5' GCA CAG TTG AAG TGA ACT TGC3' The results obtained showed that the inserts of the 5 cDNA library had an average dimension of 1.4 kb. Example 5 Conversion of the HybridZap phagic library to the pAD GALA4 plasmid library The primary library constructed in the A HybridZap 10 phage was converted to the pAD-GAL4 plasmid library by means of total excision in vivo according to the method described by the supplier of the kit adopted (Strata gene). For the amplification of the primary library, lxlO6 15 phages were used to infect the host cells XL1 Blue MRF', which, by enabling replication inside the phages, after their lysis, allowed a library consisting of about 1x10 9 phagic particles to be recovered. An aliquot of the amplified library (lxlO pfu) was 20 then incubated with 1x10 10 pfu of ExAssist helper phage and 1x10 9 of XL1 Blue MRF' cells to generate particles of phagemid containing the plasmid vector excised from the phage vector. The excess number of phage helpers and bacterial WO 00/70052 PCT/EPO0/04181 24 cells with respect to the number of phages of the li brary was used to ensure that each cell was infected both by the phage helpers and by the lambda phage, thus obtaining an effective and representative excision in 5 vivo. The incubation of the lambda phages with the phage helpers and XL1 Blue MRF' cells took place at 37'C for 15 minutes, after which 20 ml of LB medium were added and the incubation was continued at 370C for a further 10 3 hours. In order to lyse the phage particles and enable the particles of phagemid to be released and recovered, the suspension was incubated at 700C for 20 minutes and then centrifuged for 10 minutes at 500 revs. The super 15 natant (phagemid stock) was recovered and conserved at 4 0C. Cells of E.coli XLOR were incubated with 1x108 of phagemid in a ratio of 10:1, at 37'C for 15 minutes. 500 ml of LB medium containing 50 pg/ml of ampicillin 20 were then added and the incubation was continued at 370C for 3 hours. This operation allowed cells to be obtained, con taining the target library in a plasmid vector. The suspension was centrifuged for 10 minutes at WO 00/70052 PCT/EP00/04181 25 500 revs and the plasmidic DNA was isolated from the pellet according to the protocol of alkaline lysis sug gested by Sambrook, J. Et al., 1989, Cold Spring Harbor laboratory Press. 5 Example 6 Screening of the library To identify the "target" proteins which interact with the "bait" protein, the yeast strain S.cerevisiae YRG-2, containing the reporter genes his3 and lacZ, was 10 co-transformed with the DNA prepared from the bait plasmids and the DNA isolated from the target plasmid library. About 5 pLg of DNA extracted from the plasmids pBD GALA4/C18, pBD-GALA4/C18-5' and pBD-GALA4/C18-3', were 15 co-transformed, in separate co-transformation proc esses, with 10 ig of DNA extracted from the target li brary. The co-transformations were plated on SD selec tive medium (yeast nitrogenated base without aminoacids 6.7 g/l, D-sorbitol 182.2 g/l, agar 20 g/l, 100 ml of 20 the appropriate aminoacidic solution concentrated lOx and glucose 2%) without leucine aminoacids (Leu ), tryp tophan (Trp ) and histidine (His-), which allows only the growth of the yeast colonies containing both of the recombinant plasmids pBD-GALA4 (Trp ) and pAD-GAL4 (Leu WO 00/70052 PCT/EPOO/04181 26 ), and incubated at 30'C for 5 days. The yeast colonies thus obtained were transferred to Watman 3MM filter pa per and subjected to the expression test for the re porter gene lacZ, by soaking the filters in a solution 5 containing the chromogenic substrate 5-bromo-4-chloro indolyl @-D-galactoside (X-Gal) and incubating at 300C for a night. From the test of the 3 sequences of oleate 12 hydroxylase with the library, 7 yeast colonies were 10 identified, having the typical blue colour of the ac tivity of the gene lacZ, thus indicating the probable interaction of the bait protein with an unknown target protein. To verify the authenticity of the above colo nies, these were re-plated on selective medium and the 15 X-Gal test was repeated. Of the 7 colonies identified, only one reconfirmed the previous phenotype, whereas the others proved to be false positives. The positive yeast colony proved to come from the 20 co-transformation process in which the plasmid pBD GAL4/C18-5' was used, containing the 5' region of the oleate 12-hydroxylase gene. Transformation control experiments were parallelly carried out with the 4 plasmids pGal4, p53, pSV40 and WO 00/70052 PCT/EPOO/04181 27 pLaminC (Stratagene) . These plasmids were used singly or in pairs as indicated in table 1 and, on the basis of the combinations, they acted as positive or negative controls. Table 1 also indicates the results of the 5 transformation processes in which the bait protein in teracts with an unknown target protein. Table 1 Transformations SDl SD2 SD3 SD4 GROWTH 10 pGAL4 blue p53 white pSV40 white pLaminC white p53 and pSV40 blue blue 15 pLaminC and pSV40 white 0 pBDGAL4-Bait white pBDGAL4-Target white pBDGAL4-Target pBDGAL4-Bait white white blue blue 20 wherein SD1 = SD medium without Leu; SD2 = SD medium without Trp; SD3 = SD medium without Leu and Trp and SD4 = SD medium without Leu, Trp and HIs. Example 7 Verification of the interaction specificity WO 00/70052 PCT/EPOO/04181 28 To verify the interaction specificity between ole ate 12-hydroxylase and the new protein identified, co transformation experiments were carried out in yeast in which the DNA of the target plasmid, called pTargl, was 5 tested in combination with the whole gene of oleate 12 hydroxylase, with the 5' region and with the genes of the bait proteins p53 and pLaminC. To isolate the plasmid DNA pTargl from the yeast colony resulting positive from the X-GAL test, the lat 10 ter was inoculated into 2 ml of YPAD growth medium (peptone 20 g/l, yeast extract 10 g/l, adenine sulfate 40 mg/l and glucose 2%) and incubated at 300C for 2 days. To recover the plasmid DNA the cellular wall of 15 the yeast was mechanically broken using glass balls and the nucleic acids were precipitated following the pro cedure described by Sambrook, J. Et al., 1989, Cold Spring Harbor Laboratory Press. About 5 pg of the plasmid pTargl were used in a 20 co-transformation experiment with 5 p.g of DNA of the plasmid containing the whole oleate 12-hydroxylase gene (pBD-GAL4/C18), and in another experiment with 5 tg of DNA of the plasmid containing 5-terminal portion of the oleate 12-hydroxylase gene (pBD-GAL4/C18-5') . 2 yeast WO 00/70052 PCT/EPOO/04181 29 colonies were identified from the tests of the two se quences, one for each co-transformation process, which had the typical blue colour of the activity of the lacZ gene and thus indicating the interaction of the bait 5 protein with an unknown target protein. The above colonies were plated on selective SD me dium (Leu~, Trp~, His-) and tested again with X-Gal. The two colonies identified reconfirmed the previous pheno type. 10 When, on the other hand, the target protein was tested with the bait proteins p53 and pLaminC, as ex pected, none of the colonies had the blue colour with the X-Gal test. These results indicated that the new protein identified interacted specifically with oleate 15 12-hydroxylase. To confirm the presence and dimensions of the pro teins used as bait and to verify the presence and di mensions of the fragment encoding for the target pro tein, called TargH12, PCR analyses were effected on the 20 DNA extracted from the two yeast clones resulting posi tive from the expression test of the reporter gene lacZ. Specific primers for the Binding Domain (BD) re gion and for the Activation Domain (AD) region were WO 00/70052 PCT/EPOO/04181 30 used for the amplification under the conditions sug gested by the kit supplier. The sequences of the specific primers for the Ac tivation Domain were specified in example 4, whereas 5 the sequences for the Binding Domain are the following: a) 5'BD: 5'GTG CGA CAT CAT CAT CGG AAG3' b) 3'BD: 5'CCT AAG AGT CAC TTT AAA ATT3' The authenticity of the amplification products was demonstrated by the size of the expected fragments and 10 also by the hybridization analysis carried out on these. Example 8 Sequence analysis of the plasmid Targ1 The plasmid DNA of pTargl was subjected to ampli 15 fication reaction with the use of specific primers of the plasmid pAD-Gal4, 5'AD primer and 3'AD primer. The fragment produced was purified with the GeneCleanTM Kit (BIO 101 Inc. U.S.A.). About 100 ng of the DNA thus pu rified were ligated with 50 ng of the plasmid pGEM-T 20 (Promega) in 10 pl of reaction mixture, in the presence of 2 units of T4 DNA ligase, at 4'C for a night. 5 pg of the ligase mixture were used to transform competent cells of E.coli DH5a (BRL) . The transforming agents were selected on LB medium to which 50 pg/ml of WO 00/70052 PCT/EP0O/04181 31 ampicillin had been added. The plasmid DNA was extracted from 6 positive clones, i.e. showing a white colour, and sequenced with the Taq Dye Deoxy T Cycle Sequencing T M Kit (AB-PEC) , us 5 ing an ABI Prism 373A DNA Sequencer (AB-PEC). From the sequence analyses, it can be observed that the isolated gene of 762 bp (SEQ: ID Nr:4) con tains an Open reading frame (ORF) of 540 bp, preceded by 75 bp at 5' and followed by 147 bp at 3' where the 10 poly (A) tail is present. The ORF encodes a protein of 180 aminoacids (SEQ: ID Nr:5), indicated as TargH12, with a molecular weight of 19.8 kilodaltons. Example 9 Southern Blot analysis 15 To verify the identity of the protein capable of interacting with oleate 12-hydroxylase and the number of ricin copies of the gene corresponding to the insert of the plasmid pTargl, analyses were carried out on the genomic DNA of different species, isolated with the 20 method of Dellaporta et al., 1983, Plant Mol. Biol.: 1, 19-21. About 6 ptg of genomic DNA isolated respectively from: Ricinus communis, Lesquerella fendleri, Linum usitatis- WO 00/70052 PCT/EPOO/04181 32 simum, Brassica napus, Helianthus annus, Limnantes douglasii, Lycopersicon esculentum, Beta vulgaris, Zea mays, Nicotiana tabacum and Saccharomyces cerevisiae, were digested with 100 units of the enzyme EcoRI (Boe 5 hringer) in 100 p1 of reaction mixture, at 370C for 1 hour. The digestion mixtures were charged onto agarose gel at 0.8% and subjected to horizontal electrophore sis. The DNA was transferred onto nitrocellulose filter 10 (Hybond-N(+)R, Hamersham) with the Southern method (Sam brook, J. Et al., Cold Spring Harbor Laboratory Press). This filter was hybridized for a night at 650C, after a pre-hybridization of about 4 hours at 650C, in a solution containing 6XSSC, 1% SDS, 1OX Denhardt's and 15 tRNA at a concentration of 10 pg per ml of hybridiza tion solution used. The fragment of 762 bp isolated from the plasmid pTargl, marked with 3 2P (Sambrook and Fritsch, E.F. and Maniatis, T. (1989), Molecular Clon ing: A Laboratory Manual, (Cold Spring Harbor Lab., 20 Cold Spring Harbor, NY) 2 Ed. 1989), was used as probe. The filter was washed twice at 650C in 2XSSC, 0.2% SDS for 30 minutes and once in 0.2XSSC, 0.02% SDS for 20 minutes, then exposed to X-rays by autoradiography.

WO 00/70052 PCT/EPOO/04181 33 The hybridization showed a single band only on the genomic DNA isolated from R.communis. This demonstrated that the gene isolated was specific of the genome of this plant, which was present in a single copy and that 5 the result obtained was not an outcome of the system used for its identification (figure 1). Example 10 Expression analysis To effect the expression analysis of the gene en 10 coding the protein TargH12, the messenger RNA was pre pared from different organs (leaves, stem and roots) of R.communis and at different development stages of the seed (10-20-30-35-40 days after pollination, DAP). About 7 pig of each sample were charged onto aga 15 rose gel at 1.4% containing formaldehyde and subjected to an electrophoretic run. The RNA were transferred onto a nitrocellulose filter (Hybond-N (+) Hamersham) using the standard Northern blot procedure. The filter was hybridized with a probe corresponding to the frag 20 ment of 762 bp of the plasmid pTargl marked with 32 P. The reaction was carried out in a hybridization solu tion containing 6XSSC, 1%SDS, 1oX Denhardt's and 10 pg of tRNA, at 650C for a night, after a pre-hybridization of 5 hours in the same solution at 650C.

WO 00/70052 PCT/EPOO/04181 34 The filter was washed twice at 65 0 C in 2xSSC, 0.2%SDS for 20 minutes and once in 0.2XSSC, 0.02%SDS at 65*C for 20 minutes and then exposed to X-rays by auto radiography. 5 The results showed the presence of a signal, lo calized in a band of about 1 Kb (figure 2), in the sam ples corresponding to unripe seeds of 10 and 20 DAP. A similar signal was observed in the sample of RNA ex tracted from the leaves. 10 The same Northern filter was hybridized with the fragment of oleate 12-hydroxylase which begins to ex press itself in the unripe seeds at 20 DAP, where the signal is very weak, subsequently increasing its ex pression in the stages at 30, 35 and 40 DAP (figure 3). 15 No hybridization signal was observed in the samples of RNA corresponding to the leaves, stem and roots. From these results it can be deduced that the pro tein isolated with the double hybrid system most proba bly intervenes in the first stages of development of 20 the seed of R.communis, at the beginning of the synthe sis of ricinoleic acid.

WO 00/70052 PCT/EPOO/04181 35 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM Metapontum Agrobios RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT S.S. lonica 106 km. 448,2 issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY 75010 MTidentified at the bottom of this page Italid name and address of depositor I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY: MA292 CBS 101642 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above was accompanied by: a scientific description a proposed taxonemic designation (mark with a cross where applicable) III. RECEIPT AND ACCEPTANCE This International Depositary accepts the microorganism identified under I above, which received by it on 0403-9g (date dd-mm-yy of the original deposit) 1 IV. RECEIPT OF REQUEST FOR CONVERSION The microorganism identified under I above was received by this International Depositary Authority on not applicable (date dd-mm-yy of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on not applicable (date dd-mm-yy of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY Name: Centraalbureau voor Schimmelcultures Signature(s) of persons) having the power to represent the International Depositary Authorto-f auhrzdof al Address: Oosterstraat 1 P.O. Box 273 3740 AG BAARN Mrs F.B. Snippe-Claus JA S rs The Netherlands Date (dd-m-yy): 1 3-99 1 Where Rule 6.4(d) applies, such date is the date on which the status of,irternational depositary authority was acquired. Form BP/4 (sole page) CBS/9107 WO 00/70052 PCT/EPOO/04181 36 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM Metapontum Agrobios VIABILITY STATEMENT S.S. lonica 106 km. 448,2 issued pursuant to Rule 10.2 by the 75010 METAPONTO MT INTERNATIONAL DEPOSITARY AUTHORITY Italid identified on the following page name and address of the party to whom the viability statement is issued I. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM Name: Metapontum Agrobios Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: CBS 101642 Address: S.S. lonica 106 km. 448,2 75010 METAPONTO MT Date (dd-mm-yy) of the deposit or of the Italid transfer: 1 III. VIABILITY STATEMENT The viability of the microorganism identified under II above was tested on 08-03-99 2 . On that date (dd-m-yy), the said microorganism was 3 3 viable 3 3 no longer viable 1 Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer). 2 In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability test. 3 Mark with a cross the applicable box. Form BP/9 (first page) WO 00/70052 PCT/EPOO/04181 37 IV. CONDITIONS UNDER WHICH THE VIABILITY HAS BEEN PERFORMED V. INTERNATIONAL DEPOSITARY AUTHORITY Name: Centraaibureau voor Schimmelcultures Signature(s) of persons) having the power to represent the International Depositary -atoie official (s): Address: Oosterstraat 1 P.O. Box 273 3740 AG BAARN Mrs F.B. Snippe-Claus S pers The Netherlands Date (dd-zm-yy) : 11-03 4 Fill in if the information has been requested and if the results of the test were negative. Form iP/n (second and last page)

Claims (8)

1. A gene isolated from the genomic DNA of Ricinus communis characterized by the nucleotide sequence SEQ ID Nr: 4. 5

2. A recombinant expression vector comprising the gene having the nucleotide sequence SEQ ID Nr: 4.

3. The vector according to claim 2, deposited as E.coli DH5a MA292 with the deposit number CBS

101642. 10

4. A microorganism transformed with the recombinant expression vector according to claim 2.

5. Transgenic plants comprising in their cells the gene having the nucleotide sequence SEQ ID Nr: 4.

6. The transgenic plants according to claim 5, se 15 lected from Arabidopsis thaliana, Linum usitatis simum, Helianthus annus and Brassica napus.

7. Seeds obtained from the transgenic plants accord ing to claim 5.

8. A protein characterized by the aminoacid sequence 20 having the sequence SEQ ID Nr: 5.