ITMI991080A1 - GENE ISOLATED BY RICINUS COMMUNIS CODING FOR A NEW PROTEIN THAT INTERACTS WITH THE 12-HYDROXYLASE OIL ENZYME - Google Patents

Description

Description

The present invention relates to the identification and characterization of a Ricinus communis (R.communis) gene which codes for a protein capable of interacting with the oleate 12-hydroxylase enzyme which catalyzes the introduction of a hydroxyl group in the molecule of oleic acid transforming it into ricinoleic acid.

The invention also relates to means and methods for producing transgenic plants with a modified fatty acid composition.

Ricinoleic acid (12-hydroxy-9-octadecenoic) is a monohydroxylated fatty acid, the only commercial source of which is the seed oil synthesized in the endosperm of the ripe seeds of R.communis, where it represents about 90% of the hydroxylated fatty acids.

In vivo studies with radioactive tracers indicate that, in the endosperm of immature seeds of R.communis, ricinoleic acid (also known as ricinoleate) is synthesized by the direct replacement of a double bond of oleic acid with a hydroxyl group ( Morris, L.J. 1967, Biochem. Biophys. Res. Commun. 29, 311-315). This reaction is catalyzed by the oleate 12-hydroxylase enzyme whose activity appears to be associated with the endoplasmic reticulum.

Enzymatic assays indicate that the substrate for oleate 12-hydroxylase is oleic acid esterified with phosphatidylcholine or with another phospholipid; esterified ricinoleate is released from the lipid complex thanks to the intervention of a phospholipase A, specific for oxygenated fatty acids in the presence of molecular oxygen, NAD (P) H and cytochrome bs- NAD (P) H is required to reduce 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 fatty acid is then transferred, via the Kennedy pathway, to the triacylglycerol pathway where it is accumulated.

Ricinoleic acid, due to the presence of the hydroxyl group, is one of the most versatile natural products and has numerous industrial and food applications. In particular, ricinoleic acid can be used in the production of paints, nylon-11 type polymers, drugs, lubricants, cosmetics, resins and other materials.

The production of ricinoleic acid, however, is limited by the high susceptibility to climatic variations of the crops of R.communis plants and by the toxicity of ricin, an allergen present in castor beans.

The possibility of producing ricinoleic acid in plant species more tolerant to climatic variations and which do not contain toxic substances would allow a greater and simpler production and application of the acid itself.

For this purpose, recently the gene encoding the oleate 12-hydroxylase enzyme was isolated and used to transform plants such as Nicotiana tabacum, Arabidopsis thaliana, Linum usitatissimum and Brassica napus.

In all these cases, however, while observing an altered fatty acid content, the increase in ricinoleic acid was low if not zero (Broun P. E Somerville C., 1997, Plant Physiol, 113, 933-942).

It is known that in many biological processes, such as replication, transcription or metabolism, enzyme complexes intervene whose action is correlated to the cooperation of several protein subunits.

For example, evidence of the possible interaction of several proteins in desaturation processes has been obtained from studies on the activity of the enzyme toluene 2-monooxygenase isolated from the bacterium Burkholderia cepacia (Newman L.M., et al., 1995, Biochemistry, 34, 14066- 14076).

It was then hypothesized that also the hydroxylases of vegetable fatty acids can be part of a multicomponent system and that, therefore, the hydroxylase activity of the enzyme oleate 12-hydroxylase of R.communis requires the intervention of further cofactors or proteins.

A gene of R.communis has now been identified and characterized, encoding a new protein capable of interacting with the oleate 12-hydroxylase enzyme. This gene can be used in genetic transformation programs of plants containing the enzyme oleate 12-hydroxylase to promote the production of ricinoleic acid.

Accordingly, an object of the present invention is the cloned and sequenced gene which encodes a protein capable of interacting with oleate 12-hydroxylase.

A further object of the present invention is a recombinant vector with expression in host cells comprising said gene.

A further object of the present invention is a host microorganism transformed with said vector.

Another object of the present invention is transgenic plants transformed with said vector.

Further objects of the present invention will become evident from reading the description and the examples.

Brief description of the figures

Figure 1: Southern blot of genomic DNA of different species digested with the restriction enzyme EcoRI and hybridized with the 762 bp fragment of the plasmid pTargl labeled with <32> P. The hybridization signal corresponds to a single copy gene evident only in R.communis.

Figure 2: Northern blot of messenger RNA extracted at different stages of seed development (10, 20, 30, 35 and 40 DAP), from the leaves, stem and roots of the R.communi s plant. The filter was hybridized with the 762 bp fragment of the pTargl plasmid labeled <32> P. The presence of an mRNA with a molecular weight of about 1.0 Kb is observed in immature seeds at 10 DAP and 20 DAP and a larger transcript in the leaves.

Figure 3: Northern blot of messenger RNA extracted at different stages of development 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 1216 bp fragment of the 32P-labeled oleate 12-hydroxylase. The hybridization signal, of about 1.6 Kb, is present in immature seeds at 20, 30, 35 and 40 DAP.

Detailed description of the invention

The isolation of nucleotide sequences which code for proteins of interest can be carried out by operating according to known techniques.

In particular, Stratagene's "HybridZap two hybrid vector", a eukaryotic system (Saccharomyces cerevisiae that allows to identify new genes encoding proteins that interact with a known protein (Fields S. Et al.

1989, Nature, 340, 245-246). This system exploits the characteristics of the transcriptional activator GAL4 of S.cerevisiae, which regulates the expression of genes that encode enzymes involved in the metabolism of galactose.

GAL4 consists of two separable and functionally essential domains for its activity: an N-terminal domain (BD), which binds to specific DNA sequences (UAS: upstream activating sequences), and a C-terminal domain, containing acidic regions (Activation Domain, AD), which is required for transcriptional activation.

The system used allows to generate two hybrid proteins containing the functional domains of GAL4, namely the Binding Domain fused to a known protein that acts as bait, and the Activation Domain fused to unknown proteins (targets) from a library of expression.

If the known protein interacts with a target protein forming a protein-protein complex, the two functional domains of GAL4 bring themselves to optimal conditions and activate the transcription of the lac-Z reporter gene, the product of which is highlighted by a colorimetric reaction.

Total RNA was extracted from a pool of immature R.communis seeds using the "Hot-Phenol" method and polyadenylated messenger RNA (mRNA) was isolated with oligo-dT columns (Pharmacia). Subsequently the cDNA encoding the oleate 12-hydroxylase enzyme was prepared by applying the polymerase chain reaction (PCR) technique on the mRNA using as primers a pair of oligonucleotides having the sequences flanking the coding region including the start codon and stop of the translation of said gene.

Since the interaction of target proteins can occur with the entire protein under examination or with parts of it, it was decided to clone in the plasmid pBD-GAL4, which contains the sequence that codes for the Binding Domain, both the entire gene encoding for oleate 12-hydroxylase that its terminal 5 'and 3' regions.

The three DNA inserts were first amplified with the appropriate "primers" having the EcoRI restriction sites for the Forward primer and Salts for the Reverse primer, digested with the aforementioned enzymes, inserted directionally in the pBD-GALA4 vector predigested with the same enzymes and introduced into the competent Epicurian coli XLl-Blue cells.

The recombinant clones, containing the expected fragments, were characterized by restriction analysis and their identity was confirmed with the sequence reactions conducted using the Taq Dye Deoxy Terminator Cycle Sequencing kit (Perkin Elmer) and analyzed with the automatic sequencer. Subsequently, a cDNA expression library from immature seeds of R.communis was prepared in the phage vector λ HybridZap expressing hybrid proteins consisting of the Activation Domain of GALA4 and proteins of R.communis.

In practice, the polyadenylated mRNA of R.communis was used for the synthesis of double-stranded cDNA operating according to the protocols suggested by the distributor of the Kit (Stratagene).

Then the high molecular weight cDNA molecules, useful for the construction of the library, were separated from the low molecular weight ones, which represent the fraction of molecules in which the synthesis was not completed.

The high molecular weight cDNA fraction was inserted into the λ HybriZap phage vector and packed with the packaging extracts containing the phage head and tail proteins. The dimensions of the inserts present in the library produced were verified by PCR and amplified with a pair of primers specific for the pAD-GAL4 vector. The results obtained showed that the fragments of the cDNA library had an average size of 1.4 Kb.

After amplification, the library in the lambda phage was converted to a plasmid library by excision in vivo.

Subsequently the library was multiplied in the E. coli XLOR strain and the plasmid DNA was extracted and co-transformed, in separate cotransformation events, with the DNA extracted from the bait plasmids containing the whole gene and parts of the oleate 12-hydroxylase, using yeast cells (YRG-2 strain), having the his3 and lacZ reporter genes.

From the assay of the three oleate 12-hydroxylase constructs with the expression library, colonies of yeast were identified that presented the blue color typical of lacZ gene activity and, therefore, demonstrating the probable interaction of the bait protein with an unknown target protein. . Subsequent analyzes on these colonies allowed the identification of a "positive" co-transformed yeast clone, which activated the transcription of both reporter genes. This indicated the interaction between the N-terminal region of oleate 12-hydroxylase and an unknown Target protein.

The plasmid isolated from said positive yeast clone was indicated with the initials pTargl.

The specificity of interaction between oleate 12-hydroxylase and the newly identified protein was confirmed through co-transformation experiments in S.cerevisiae YRG-2 yeast cells with the decoy plasmids containing, respectively, the entire oleate 12- gene. hydroxylase and the terminal 5 'portion of this gene. From the assays of the two constructs, two colonies of yeast were identified, one for each co-transformation event, which presented the blue color typical of lacZ gene activity.

To confirm the presence and size of the bait proteins and to verify the presence and size of the gene encoding the Targl2 protein identified, PCR analyzes were performed on the DNA extracted from the yeast clones that tested positive for the test. expression of the lacZ reporter gene, using specific primers for the Binding Domain region and for the activation domain region.

The authenticity of the amplification products obtained was demonstrated not only by the size of the expected fragments, but also by the hybridization analysis carried out on them.

In addition, the plasmid DNA of pTargl was isolated from the yeast colony and the cDNA insert was purified with the "Doublé GeneClean" kit (BIO 101 Ine., USA). The purified fragment was then cloned into the pGEM-T vector (Promega) and then introduced into competent E. coli DH5a cells.

The plasmid DNA isolated from the recombinant clones was subjected to sequence analysis with the Taq Dye Deoxy Termiantor Cycle Sequencing Kit (Perkin Elmer), using the ABI 373A automatic sequencer (Perkin Elmer).

From the sequence analyzes conducted on the DNA of the pTargl plasmid insert, it is observed that the isolated fragment has a size of 762 bp and contains an Open Reading Frame (ORF) of 540 bp preceded by 75 bp at 5 'and followed by 147 bp at 3 '. The ORF encodes a protein of 180 amino acids with a molecular weight of 19.8 KDa.

The nucleotide and amino acid sequences were compared with the sequences available on databases through FASTA and BLAST analyzes, and no homologous sequences were found, indicating the uniqueness of the R.communis DNA stretch and the uniqueness of the identified protein.

To verify the identity of the protein capable of interacting with the oleate 12-hydroxylase, analyzes were carried out on the genomic DNA of different species. The results showed the presence of a signal only in R.communis. This shows that the isolated gene is specific to the R.communis genome and not an artifact of the system used for its detection.

Furthermore, expression analyzes were carried out on the messenger RNA extracted at different stages of seed development (10, 20, 30, 35, and 40 days after pollination), from the leaves, stem and roots of the R.communis

The results of hybridization of Northern blot with the <32> P-labeled pTargl fragment showed gene expression in immature leaves and seeds at 10 and 20 days after pollination.

The same filter was hybridized with the fragment of oleate 12-hydroxylase which begins to express itself in immature seeds at 20 DAP, where the signal is very weak, and then increases its expression in stages at 30, 35 and 40 DAP. No hybridization signal, as expected, was detected in the RNA samples corresponding to leaves, stem and roots.

On the basis of these results, it can be concluded that, with great probability, the new protein intervenes in the early stages of development of the R.communis seed, when the synthesis of ricinoleic acid is at the beginning, carrying out a regulatory action. The gene of the present invention can be cloned into a plant expression vector, placing it under the control of suitable regulatory sequences (promoter and terminator).

vectors suitable for the purposes of the present invention are for example those derived from the Ti plasmid of Agrobacterium tumefaciens as described by Bevan M., (1984), Nucleic Acid Research 12: 8711-8721.

These vectors are used to transform plants using conventional methods. Preferably, the method described by G. An et al. (Binary vectors, Plant Molecular Biology Manual A3, Kluwer Academic, Dordrecht, pp 1-19, 1988) is used, which is based on the ability of Agrobacterium tumefaciens to transfer part of its own DNA in plant cells.

The pTargl plasmid containing the gene of the present invention was deposited as E.coli DH5a / MA292 at the CentraalBureau Voor Schimmelcultures where it received the CBS filing number 101642.

The following examples, which have the sole purpose of describing the present invention in greater detail, must in no way be interpreted as a limitation to the purposes thereof.

Example 1

RNA isolation

Total RNA was extracted from a pool of immature seeds (10-40 days after pollination) of Ricinus communis by the "Hot-Phenol" method described by Shirzadegan M. et al. (1991), Nucl. Acids Res.:19,6055, to which some changes have been made.

Briefly, 2 g of plant material was crushed in liquid nitrogen and then suspended in 6 ml of extraction buffer (0.1 M LiCl, 0.1 M Tris-HCl pH 7.6, 0.01 M EDTA, 1 % Sodiumdodecyl sulfate (SDS), phenol) preheated to 80 ° C. The suspension was incubated at 80 ° C for 5 minutes and then added with 3 ml of a chloroform / isoamyl mixture (24: 1, v / v). The sample was mixed on vortex and subsequently centrifuged at 12,000 rpm, at 4 ° C for 15 minutes. The aqueous phase was recovered, subjected to a further extraction cycle with phenol / chloroform / isoamyl (25: 24: 1) and centrifuged under the same conditions reported above. The supernatant was recovered and the RNA precipitated by adding a volume of 4 M LiCl. The sample was incubated at -20 ° C overnight and then centrifuged at 13,000 rpm, at 4 ° C for 30 minutes.

The RNA pellet was resuspended in water, transferred into microcentrifuge tubes and precipitated by adding 4 M LiCl and 0.2 volumes of 0.5 M EDTA. After centrifugation at 13,000 rpm, at 4 ° C for 30 minutes, the pellet was again suspended in water and precipitated with 0.1 volumes of 5 M NaCl and 2.5 volumes of 100% ethanol. After centrifugation at 15,000 rpm, at 4 ° C for 30 minutes, the pellet was recovered, washed twice with 70% ethanol, dried and resuspended in water. Example 2

Isolation of the cDNA coding for oleate 12-hydroxylase

The polyadenylated messenger RNA (polyA-RNA) was prepared from the total RNA obtained in Example 1, by the use of oligo-dT columns (Pharmacia) according to the instructions provided by the distributor.

Then 3.5 μg of polyadenylated messenger RNA were used for the synthesis of double-stranded cDNA using the Kit distributed by PHARMACIA, operating under the experimental conditions suggested by the kit supplier.

On the basis of the oleate 12-hydroxylase sequence deposited in the database (GenBank, AC U22378) the following oligonucleotides were synthesized

(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 oligonucletides, which correspond to the regions flanking the enzyme-coding portion comprising the translation start and end codon, were used as primers for the isolation of the fragment corresponding to oleate 12-hydroxylase by the polymerase chain reaction (PCR) technique.

Amplification was carried out in a DNA Thermal Cycler 480 apparatus (Perkin Elmer Cetus) using a reaction mix (25 μΐ) containing 6 μΐ of double stranded cDNA, 10 mM Tris HC1 pH 8.3, 1.5 mM MgC12 , 50 mM KC1, 2.5 μΜ of each primer, 0.1 mM dNTP and 2.5 Units of Taq polymerase (Boheringer).

After a first denaturation cycle for 5 minutes at 95 ° C, the reaction continued with the following cycles: 1 minute at 94 ° C (denaturation)

1 minute at 56 ° C (pairing)

2 minutes at 72 ° C (elongation)

for a total of 35 cycles, followed by 10 minutes at 72 ° C (final extension).

The amplification product, corresponding to a fragment of about 1200 base pairs, was separated on 1.0% agarose gel, the DNA band of interest was recovered and purified with the GeneClean ™ kit (BIO 101 Ine , USA). Approximately 100 ng of the DNA thus isolated was ligated to 50 ng of plasmid pGEM-T (Promega) in 10 μΐ of reaction mixture, in the presence of 2 units of T4 DNA ligase, at 4 ° C overnight.

5 μΐ of said mixture were used to transform competent E.coli DH5a (BRL) cells. The transformants were selected on plates of LB medium (NaCl 10 g / 1, Yeast extract 5 g / 1, Bacto-tryptone 10 g / 1 and agar 20 g / 1) containing 50 pg / ml of ampicillin.

The plasmid DNA extracted from 6 positive clones was subjected to sequence analysis to verify the nucleotide correspondence with the oleate 12-hydroxylase gene isolated by Van de Loo F. Et al., 1995, PNAS, 92, 6743-6747. Reactions and sequence analyzes were conducted with the Taq Dye Deoxy Terminator Cycle Sequencing ™ (AB-PEC) kit using the ABI Prism 373A DNA Sequencer (AB-PEC).

One of the plasmids analyzed, containing a DNA fragment similar to the published sequence SEQ: ID No 1, was named pC18-MA.

Example 3

Construction of "bait" vectors (pBD-GAL4)

Since the interaction of target proteins can occur with the entire protein under examination (bait) or parts of it, it was decided to clone in the plasmid pBD-GAL4 (Stratagene) both the fragment corresponding to the entire gene coding for oleate 12-hydroxylase, which the fragments corresponding to the 5 'and 3' regions of said gene.

For this purpose, four oligonucleotides have been synthesized, of which the Forward primers (marked with F) have the EcoRI restriction site and the Reverse primers (marked with R) the Sali site. The nucleotide sequences of the primers are as follows:

(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) e

(d) 5'GAA TTC AAT GTC TCT GGT AGA C 3 '(GalHydi-F)

The pair of HydGal-F and HydGal-R primers was used to amplify the entire oleate 12-hydroxylase gene.

The HydGal-F / GalHydi-R and HydGal-R / GalHydi-F pairs were used to amplify, respectively, the 5 'region of 624 bp (SEQ: ID NO: 2) and the 3' region of 633 bp (SEQ : ID NO: 3) of the oleate 12-hydroxylase gene.

The amplifications with the aforementioned base pairs were carried out on 20 ng of the previously cloned and sequenced oleate 12-hydroxylase fragment.

The amplification products having the expected size were digested with 10 units of the restriction enzymes EcoRI and Salì (Boheringher), separated on 1% agarose gel and the DNA fragments of interest were then recovered and purified with the kit. GeneClean ™ <BIO 101 Ine.).

About 100 ng of each fragment were ligated, separately, with the pBD-GAL4 plasmid linearized with the EcoRI and Sali enzymes, in 10 ml of reaction mixture, in the presence of 2 units of T4 DNA ligase, at 4 ° C for a night. The ligase mixtures were used to transform competent cells of Epicurian coli XL1 Blue (Stratagene). The recombinant clones were selected on plates of LB medium supplemented with 30 µg / ml of chloramphenicol. The following recombinant "bait" plasmids have been identified that contain a fragment that constitutes the Gal4 Binding Domain fused to:

(a) the entire gene of oleate 12 hydroxylase (pBD-GALA4 / C1B);

(b) the 5-terminal region of the oleate 12 hydroxylase gene (pBD-GALA4 / C18-5); And

(c) the 3-terminal region of the oleate 12 hydroxylase gene (pBD-GALA4 / C18-3).

The plasmids were characterized by restriction analysis and their identity was confirmed by performing sequence reactions with the Taq Dye Deoxy Terminator Cycle sequencing kit (Perkin Elmer) and analyzed with the ABI 373A automatic sequencer (Perkin Elmer).

Example 4

Building the target library

The phage vector HybridZap was used for the construction of a "target" library, consisting of hybrid proteins consisting of the activation domain of GALA4 and proteins of immature seeds of R.communis, the phage vector HybridZap was used. The experimental conditions used were those suggested by the kit supplier (Stratagene, HybridZap ™ Two-Hybrid cDNA gapack cloning kit, catalog number: 235612).

Approximately 5 μg of R.communis polyadenylated mRNA was used for the synthesis of double-stranded cDNA.

Then, the ends of the cDNA molecules were blunted by the action of the Pfu DNA polymerase (Stratagene), added with 3.6 μg of a linker having the EcoRI restriction site and subjected to digestion with the Xhol enzyme (120 units), the site of which is present in the polydT primer used for the synthesis of the first strand of cDNA. In this way, molecules originated having the EcoRI site at one end and the Xhol site at the other.

Subsequently the cDNA sample was passed on a Sephacryl S-500 column equilibrated in 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 100 mM NaCl, and subjected to centrifugation for 2 minutes at 400 rpm.

Three fractions were recovered whose molecular weight was verified by electrophoresis on 5% non-denaturing polyacrylamide gel (Sambrook, J. Et al., (1989), Cold Spring Harbor Laboratory Press).

About 100 ng of the first fraction of cDNA, corresponding to the high molecular weight one, was ligated with 1 μg of the phage vector λ HybridZap predigested with the enzymes EcoRI and Xhol and packed with the packaging extracts containing the proteins for the head and for the phage's tail. The total amount of phage particles obtained from the in vitro packaging was determined by plating aliquots with the XL1-Blue MRF 'strain.

The primary library obtained contains a total of 1.4 x 10 <6> plaque forming units (pfu) per μg of arms of the ligated vector and 97% of them contain the DNA insert. The size of the library produced was verified by subjecting 20 phage plaques to PCR reaction, chosen randomly, and amplified with the pair of primers specific 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 cDNA library inserts had an average size of 1.4 kb.

Example 5

Conversion of the HybridZap phage library to the pAD-GALA4 plasmid library

The primary library built in the λ HybridZap phage was converted into a plasmid library pAD-GAL4 by total excision in vivo according to the method described by the supplier of the kit used (Stratagene).

For the amplification of the primary library, IxlO6 phages were used to infect the XL1 Blue MRF 'host cells, which, allowing the replication of the phages within them, allowed, following their lysis, the recovery of a library consisting of about IxlO9 phage particles.

An aliquot of the amplified library (IxlO8 pfu) was then incubated with IxlO10 pfu of ExAssist helper phage and IxlO9 XL1 Blue MRF cells to generate phagemid particles containing the excised plasmid vector from the phage one.

The excess number of helper phage and bacterial cells compared to the number of phage in the library was used to ensure that each cell was infected with both the phage helper and the lambda phage for efficient and representative excision in vivo.

The incubation of the lambda phages with the phage helpers and the XL1 Blue MRF cells is at 37 ° C for 15 minutes, after which 20 ml of LB medium were added and the incubation continued at 37 ° C for another 3 hours.

To use the phage particles and allow the release and recovery of the phagemid particles, the suspension was incubated at 70 ° C for 20 minutes and then centrifuged for 10 minutes at 500xg. The supernatant {phagemid stock) was recovered and stored at 4 ° C. E. Coli XLOR cells were incubated with 1x10s phagemid in a 10: 1 ratio, at 37 ° C for 15 minutes. Then 500 ml of LB medium containing 50 4g / ml of ampicillin were added and the incubation continued at 37 ° C for 3 hours.

This operation allowed the obtaining of cells containing the target library in a plasmid vector.

The suspension was centrifuged for 10 minutes at 500xg and plasmid DNA was isolated from the pellet according to the alkaline lysis protocol suggested by Sambrook, J. Et al., 1989, Cold Spring Harbor laboratory Press.

Example 6

Library screening

To identify the "target" proteins that interact with the "bait" protein, the S.cerevisiae YRG-2 yeast strain, containing the his3 and lacZ reporter genes, was co-transformed with the DNA prepared from the decoy plasmids and the DNA isolated from the target plasmid library.

Approximately 5 μg of DNA extracted from pBD-GALA4 / C18, pBD-GALA4 / C18-5 'and pBD-GALA4 / CI8-3' plasmids, were co-transformed, in separate cotransformation events, with 10 μg of extracted DNA from the target library. Co-transformations were plated on SD selective medium (amino acid-free yeast nitrogen base 6.7 g / 1, D-sorbitol 182.2 g / 1, agar 20 g / 1, 100 ml of the appropriate concentrated amino acid solution 10x and glucose 2%) free from the amino acids leucine (Leu-), tryptophan (Trp <">) and histidine (His <~>), which allows growth only of yeast colonies containing both recombinant pBD-GALA4 plasmids (Trp <" >) and pAD-GAL4 (Leu <'>), and incubated at 30 ° C for 5 days. The yeast colonies thus obtained were transferred to Watman 3MM filter paper and subjected to the expression assay for the lacZ reporter gene, by soaking the same filters in a solution containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal) and incubating at 30 ° C overnight.

From the assay of the 3 oleate 12-hydroxylase constructs with the expression library, 7 colonies of yeast were identified which showed the blue color typical of lacZ gene activity and therefore indicating the probable interaction of the bait protein with an unknown target protein. To verify the authenticity of the aforementioned colonies, they were replated on selective medium and the X-Gal assay was repeated.

Of the 7 colonies identified, only one reconfirmed the previous phenotype, while the others were found to be false positives.

The positive yeast colony resulted from the co-transformation event in which the pBD-GAL4 / C18-5 'plasmid, containing the 5' region of the oleate 12-hydroxylase gene, was used.

In parallel, transformation control experiments were performed with the 4 plasmids pGal4, p53, pSV40 and pLaminC (Stratagene). These plasmids were used singly or in pairs as reported in table 1 and, according to the combinations, they functioned as positive or negative controls. Table 1 also shows the results of the transformation events in which the bait protein interacts with an unknown target protein.

Table 1

where SDÌ = SD medium without Leu; SD2 = Trp-free SD medium; SD3 ~ SD medium devoid of Leu and Trp and SD4 = SD medium devoid of Leu, Trp and HIs;

Example 7

Verification of interaction specificity

To verify the specificity of interaction between oleate 12-hydroxylase and the newly identified protein, cotransformation experiments were conducted in yeast in which the DNA of the target plasmid, called pTargl, was tested in combination with the entire gene of the oleate 12-hydroxylase, with the 5 'region and with the genes of the bait proteins p53 and pLaminC.

To isolate pTargl plasmid DNA from the yeast colony which tested positive in the X-GAL assay, the latter was inoculated in 2 ml of YPAD growth medium (20 g / 1 peptone, 10 g / 1 yeast extract, 40 adenine sulfate mg / 1 and glucose 2%) and incubated at 30 ° C for 2 days.

For the recovery of the plasmid DNA, the yeast cell wall was mechanically broken using glass beads and the nucleic acids were precipitated following the procedure described by Sambrook, J. Et al., 1989, Cold Spring Harbor Laboratory Press.

About 54g of the pTargl plasmid was used in a co-transformation experiment with _5 μg of plasmid DNA containing the entire oleate 12-hydroxylase gene (pBD-GAL4 / C18), and in another experiment with 5 μg of DNA from the plasmid. plasmid containing the 5-terminal portion of the oleate 12-hydroxylase gene (pBD-GAL4 / C18-5 '). From the assays of the two constructs, 2 colonies of yeast were identified, one for each cotransformation event, which presented the blue color typical of the lacZ gene activity and therefore indicating the interaction of the bait protein with an unknown target protein.

The above colonies were plated on SD selective medium (Leu <">, Trp <~>, His <”>) and tested again with X-Gal. The two colonies identified reconfirmed the previous phenotype.

On the other hand, when the target protein was tested with the bait proteins p53 and pLaminC, as expected, no colony showed blue staining in the X-Gal assay. These results indicated that the newly identified protein interacted specifically with oleate 12-hydroxylase.

To confirm the presence and size of the proteins used as bait and to verify the presence and size of the fragment coding for the target protein, called TargH12, PCR analyzes were carried out on the DNA extracted from the two yeast clones tested positive for the assay. expression of the lacZ reporter gene.

Specific primers for the Binding Domain (BD) region and for the Activation Domain (AD) region were used for amplification under the conditions suggested by the kit supplier.

The primer sequences specific for the activation domain were reported in Example 4, while the sequences for the Binding Domain are the following:

^ a) 5'BD: 5'GTG CGA CAT CAT CAT CGG AAG3 <1>

b) 3'BD: 5'CCT AAG AGT CAC TTT AAA ATT3 '

The authenticity of the amplification products was demonstrated not only by the size of the expected fragments but also by the hybridization analysis conducted on them.

Example 8

Sequence analysis of the Targl plasmid

The plasmid DNA of pTargl was subjected to amplification reaction using pAD-GAL4 specific plasmid primers, 5'AD primer and 3'AD primer. The fragment produced was purified with the GeneClean ™ Kit (BIO 10 Ine. USA). Approximately 100 ng of the purified DNA was ligated with 50 ng of pGEM-T plasmid (Promega) in 10 μΐ of reaction mixture, in the presence of 2 units of T4 DNA ligase, at 4 ° C overnight.

5 μΐ of the ligase mixture was used to transform competent E. coli DH5a (BRL) cells. The transformants were selected on LB medium supplemented with 50 pg / ml of ampicillin.

Plasmid DNA was extracted from 6 positive clones, ie showing white staining, and sequenced with the Taq Dye Deoxy T Cycle Sequencing ™ Kit (AB-PEC), using the ABI Prism 373A DNA Sequencer (AB-PEC).

From the sequence analysis it is observed that the isolated gene of 762 bp (SEQ: ID NO: 4) contains an Open reading frame (ORF) of 540 bp, preceded by 75 bp at 5 'and followed by 147 bp at 3' where it is present the tail of poly (A). The ORF encodes a protein of 180 amino acids (SEQ: ID NO: 5), designated TargHl2, with a molecular weight of 19.8 kilodaltons.

Example 9

Souther Blot Analysis

To verify the identity of the protein capable of interacting with the oleate 12-hydroxylase and the number of copies in castor of the gene corresponding to the insert of the plasmid pTargl, analyzes were carried out on the genomic DNA of different species, isolated with the method Dellaporta et al., 1983, Plant Mol. Biol.: 1.19-21.

Approximately 6 μg of genomic DNA isolated respectively from:

Ricinus communis, Lesquerella_ fendleri, Linum usitatissimum, Brassica napus, Helianthus annuus, Limnantes douglasii, Lycopersicon esculentum, Beta vulgaris, Zea mays, Nicotiana tabacum and Saccharomyces cerevisiae, were digested with 100 units of the EcoRI enzyme reaction mixture, at 37 ° C for 1 hour.

The digestion mixes were loaded onto 0.8% agarose gel and subjected to horizontal electrophoresis. The DNA was transferred to a nitrocellulose filter (Hybond-N (+) Hamersham) by the Southern method (Sambrook, J. Et al. Cold Spring Harbor Laboratory Press).

This filter was hybridized overnight at 65 ° C, after a pre-hybridization of approximately 4 hours at 65 ° C, in a solution containing 6XSSC, 1% SDS, 10X Denhardt's and tRNA at a concentration of 10 μg per ml of solution. of hybridization used. The 762 bp fragment isolated from the plasmid pTargl, labeled with <32> P (Sambrook and Fritsch, E.F. and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Lab., Cold Spring Harbor, NY) 2nd Ed. 1989).

The filter was washed at 65 ° C 2 times in 2XSSC, 0.2% SDS for 30 minutes and once in 0.2XSSC, 0.02% SDS for 20 minutes, then exposed to X-rays for autoradiography.

Hybridization revealed a single band only on the genomic DNA isolated from R.communis. This showed that the isolated gene was specific to the genome of this plant, that it was present in a single copy and that the result obtained was not an artifact of the system used for its identification (Figure 1).

Example 10

Expression analysis

To carry out the expression analysis of the gene encoding the TargH12 protein, messenger RNA was prepared from different organs (leaves, stem and roots) of R.communis and at different stages of seed development (10-20-30-35 ^ 40 days after pollination, DAP).

Approximately 7µg of each sample were loaded onto 1.4% agarose gel containing formaldehyde and subjected to electrophoretic running. The RNAs were transferred to a nitrocellulose filter (Hybond-N (+) <R> Hamersham) using standard Northern blot procedures. The filter was hybridized with a probe corresponding to the 762 bp fragment of the plasmid pTargl labeled with <32> P. The reaction was carried out in a hybridization solution containing 6XSSC, 1% SDS, 10X Denhardt's and 10 μg of tRNA, at 65 ° C overnight, after 5 hours pre-hybridization in the same solution at 65 ° C.

The filter was washed at 65 ° C 2 times in 2xSSC, 02% SDS for 20 minutes and once in 0.2XSSC, 0.02% SDS at 65 ° C for 20 minutes and then exposed to X-rays for autoradiography.

The results showed the presence of a signal, localized in a band of about 1 Kb (figure 2), in the samples corresponding to the immature seeds of 10 and 20 DAP. A similar signal was observed in the RNA sample extracted from the leaves.

The same Northern filter was hybridized with the oleate fragment 12-hydroxylase which begins to express itself in immature seeds at 20 DAP, where the signal is very weak, and then increases its expression in stages at 30, 35 and 40 DAP (figure 3). No hybridization signal was detected in the RNA samples corresponding to leaves, stem and roots.

From these results it can be deduced that, in all probability, the protein isolated with the double hybrid system intervenes in the early development stages of the R.communis seed, when the synthesis of ricinoleic acid begins.

SEQUENCE LISTING

NUMBER OF SEQUENCE5: 15

(1) INFORMATION FOR SEQ ID NO: l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGHT: 1216 base pairs

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION:

(1) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGHT: 180 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:

Claims (1)

CLAIMS Gene isolated from Ricinus conununis genomic DNA characterized by the nucleotide sequence SEQ ID No: 4 2. An expression recombinant vector comprising the gene having the nucleotide sequence SEQ ID No: 4. The vector according to claim 2, filed as E coli DH5a MA292 with filing number CBS 101642. 4. A microorganism transformed with the recombinant vector expressing claim 2. 5. Transgenic plants comprising in their cells the gene having the nucleotide sequence SEQ ID No: 4 6. The transgenic plants of claim 5, selected from Arabidopsis thaliana, Linum usitatissimum, Helianthus annus and Brassica napus. 7. Seeds obtained from transgenic plants of claim 5. 8. A protein characterized by the amino acid sequence having the sequence SEQ ID No: 5.