WO2003002727A2

WO2003002727A2 - Hammerhead ribozyme specific for the stearoyl-acp desaturrase of different oleaginous plants

Info

Publication number: WO2003002727A2
Application number: PCT/EP2002/006585
Authority: WO
Inventors: Caterina D'ambrosio; Adriana Lucia Stigliani; Ivana Marino; Leonarda Salfi; Francesco Cellini
Original assignee: Metapontum Agrobios SCARL
Current assignee: Metapontum Agrobios SCARL
Priority date: 2001-06-28
Filing date: 2002-06-13
Publication date: 2003-01-09
Anticipated expiration: 2003-12-28
Also published as: AU2002345041A1; ITMI20011364A0; ITMI20011364A1; WO2003002727A3

Abstract

A Hammerhead ribozyme is described, which is capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme in different oleaginous plants, a vector containing the sequence which encodes said ribozyme and its use for the production of transgenic plants with an improved content of stearic acid.

Description

HAMMERHEAD RIBOZYME SPECIFIC FOR THE STEAROYL-ACP DESATURASE OF DIFFERENT OLEAGINOUS PLANTS

The present invention relates to a Hammerhead ribozyme capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme (also called Δ9 desaturase) of different oleaginous plants, a vector containing the sequence which encodes said ribozyme and its use for the production of transgenic plants with an improved con- tent of stearic acid.

In nature lipids consist of saturated fatty acids, such as palmitic and stearic acid, and mono- and poly- unsaturated fatty acids, such as oleic acid, linoleic acid and linolenic acid. Their characterization is linked to the percentage with which each of these fatty acids is present. Lipids which are solid at room temperatures are generally defined as fatty and are characterized by a high percentage of saturated fatty acids .

These acids consist of linear molecules which tend to form a "stacked" structure responsible for their high melt- ing point . The reduced presence of double bonds, moreover, gives these acids a greater resistance to high temperatures and photo-oxidative processes. Animal fats are the main source of saturated fatty acids. Lipids with a high percentage of mono- and poly- unsaturated fatty acids are, on the other hand, characterized by a lower melting point directly correlated with the unsaturation degree of the fatty acids of which they consist. They are liquid at room temperature and are commonly called oils. The main natural source of mono- and poly- unsaturated fatty acids consists of vegetable oils.

Both fats and oils are important for the transformation industry, even if their destination of use is different due to the different physico-chemical characteristics described above.

More specifically, the use of fats is particularly requested in the confectionery industry and in all food preparation processes in which fats are subjected to high temperatures . Oils are mainly used in the food industry and, those with a high content of poly-unsaturated fatty acids, mainly in the cosmetic, soap, detergent, lubricant, paint, plastic industries, etc.

Over the years, fats deriving from animals have been gradually substituted by vegetable oils as the latter do not contain cholesterol which, on the contrary, is present in the membranes of animal cells .

Oils of a vegetable origin currently form about 85% of the production of food oils and fats and represent an es- sential element in human nutrition as they provide up to 25% of caloric buildup (Broun P. et al . , 1999, Genetic Engineering of plant lipids. Ann. Rev. Nutr. 19:197-216).

As these oils are rich in poly-unsaturated fatty acids, it was necessary to subject them to a catalytic hydro- genation process to reduce the double bonds.

This process allows the melting point of the oil to be increased and gives a better aroma and greater resistance to oxidative reactions, to which fatty acids, and particularly linolenic acid, are susceptible. The oil thus modified is used for the production of margarines which are often used as analogs of butter in the confectionery and food industries .

Catalytic hydrogenation processes, however, not only have an economic repercussion but also cause the formation of trans-isomers of fatty acids which, when digested, cause an increase in the cholesterol level in the blood.

At times, this process is insufficient and it is also necessary to add other oils with a high content of saturated fatty acids, such as palm oil, and other chemical substances having the function of preventing the formation of crystals at low temperatures.

New approaches of genetic engineering have been proposed for overcoming these drawbacks, to modify the composition of vegetable oils in order to optimize them for the requirements of the human diet .

These systems are based on the partial or total inhibition of the gene expression which encodes the desaturases involved in the metabolic path of fatty acids by means of the antisense technique (U.S. Pat. 5,850,026), of co- suppression (Knutzon DS . , et al . , Proc. Natl. Acad. Sci. USA, 1992, 89:2624-28), or of the use of multimeric ribozymes (Merlo A.O., et al . , The Plant Cell., 1998, 10:603- 21) .

As far as the above inhibition strategies are con- cerned, the use of ribozymes has advantages due to their catalytic mechanism which requires only a few molecules for being effective.

The modulation of the expression of an endogenous gene, in fact, in the case of the antisense strategy or co- suppression, requires an interaction of the stoichiometric type between transgene and the target sequence.

This condition is no longer necessary when ribozymes are used, as a catalytic RNA is theoretically capable of cutting several molecules of the RNA target. The use of ri- bozymes as transgenes, moreover, reduces the probability of the creation of suppression phenomena often associated with sense or antisense construct transformation. This is probably due to the reduced dimension of the transgene and to the lower sequence homology with endogenous genes. Ribozymes are circular RNA capable of catalyzing their own fragmentation in the absence of proteins and in correspondence with particular nucleotidic sequences.

In particular, Hammerhead ribozymes (Haseloff et al . , U.S. Patent 6,127,114) represent a specific group of ribo- zymes and have the catalytic domain structure characterized by the presence of 3 helixes (I, II, III) of varying lengths (Figure 1) .

These ribozymes consist of :

- a sequence of 22 nucleotides which form the catalytic do- main; and

- two regions flanking said catalytic domain which are complementary to the RNA target . These sequences allow the RNA target to be aligned with the catalytic domain and give the reaction specificity. All the ribozymes so far designed are characterized by having a high specificity with respect to the RNA target . This specificity is conferred to the ribozyme by the perfect complementarity of the two helixes flanking the catalytic domain with the RNA target . It has now been found that, by using a suitably de- signed single ribozyme of the Hammerhead type, it is possible to modulate the gene expression which encodes the stearoyl-ACP desaturase enzyme of different oleaginous plants . In accordance with this, an objective of the present invention relates to a Hammerhead ribozyme capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme of different oleaginous plants characterized in that the two regions flanking the catalytic site are not perfectly complementary with any of the target sequences .

A further objective of the present invention relates to a DNA sequence which encodes said ribozyme.

Another objective of the present invention relates to an expression vector in plants containing the DNA sequence which encodes said ribozyme.

Yet another objective of the present invention relates to a method for increasing the content of stearic acid in different oleaginous plants which uses said ribozyme. An additional objective of the present invention relates to transgenic plants transformed with said ribozyme.

Other objectives of the present invention will appear evident from the following description and examples. Brief description of the figures Figure 1 : it is reprted the secondary structure of a ribo- zy e of the Hammerhead type.

The central box encloses the catalytic domain of the ribozyme (helix II) ; the two side boxes form, from left to right, helix III and helix I of the ribozyme. The Xs indi- cate the nucleotides of the target sequence which are paired with the two helixes I and III of the ribozyme. The Y present on the target sequence can be a G or an A depending on the target sequence on which the ribozyme has been designed (see Figure 2) . This nucleotidic base associated with the U and C forms the cleavage triplet indicated in bold letters.

Figure 2 : this indicates a comparison between the target sequence defined in the present invention and the target sequences of some oleaginous plants . The central ATC in bold letters and underlined, represent the cleavage triplet; the bases in bold letters, indicated in the sequences of the different species, are the bases different from the target sequence. Figure 3 : indicates the photo of the gel relating to the catalytic test on the transcript of the stearoyl-ACP desaturase enzymes of various species.

Line 1: Flax; Line 2: Flax + ribozyme; Line 3: Rape; Line 4: Rape + ribozyme; Line 5: Sad 6 (Sunflower); Line 6: Sad 6 (Sunflower) + ribozyme; Line 7: Sad 17 (Sunflower) ; Line 8: Sad 17 (Sunflower) + ribozyme; Line 9: Olive; Line 10: Olive + ribozyme; Line 11: Castor oil plant; Line 12: Castor oil plant + ribozyme.

In Lines 1, 3, 5, 7, 9 and 11, there is a single band which relates to the marked gene transcript; whereas in Lines 2, 4, 6, 8, 10 and 12 the two bands produced by the cutting of the target gene on the part of the ribozyme, can be observed.

Figure 4 : indicates the photo of the gel relating to the catalytic test on the transcript of the stearoyl-ACP de- saturase enzymes of various species.

Line 1: Sad 17 (Sunflower) + ribozyme; Line 2: Sad 17 (Sunflower); Line 3: Sad 6 (Sunflower) + ribozyme; Line 4: Sad 6 (Sunflower); Line 5: Rape + ribozyme; Line 6: Rape; Line 7: Flax + ribozyme; Line 8: Flax. Figure 5 : indicates the photo of the gel relating to the catalytic test on the transcript of the stearoyl-ACP desaturase enzymes of various species.

Line 1: Olive + ribozyme; Line 2: Olive; Line 3: Castor oil plant + ribozyme; Line 4: Castor oil plant; Line 5: Rape + ribozyme; Line 6: Rape; Line 7: Flax + ribozyme; Line 8: Flax; Line 9: Sad 17 (Sunflower) + ribozyme; Line 10: Sad 17 (Sunflower) . Detailed description of the invention

In the seeds of oleaginous plants, the synthesis of fatty acids seem to take place in the plastids and these acids are subsequently conveyed into the endoplasmic re- ticulum, where they are used for the formation of triglyc- erides .

The first product in the synthesis process of these acids is palmitate (16:0), whose carbon atom chain is lengthened to stearate (18:0) on which the stearoyl-ACP desaturase enzyme (commonly called Δ9 desaturase) acts, when it is still inside the plastid.

This enzyme adds a double cis bond in position 9/10 of the chain, converting the stearoyl-ACP into oleoyl-ACP (stearate to oleate) .

In order to construct a ribozyme capable of interacting with a wider range of target RNA having a region with a high homology among each other, the sequences of the Δ9 de- saturase enzymes of Linum usitatissimum, Brassica napus,

Ricinus comunis, Helianthus annuus, Carthamus tinctorius,

Simmondsia chinensis, Plea europaea and Sesamum indicum, were taken into consideration.

The stearoyl-ACP desaturase enzymes of these plants were aligned and compared and a cleavage site (ATC) was identified in a highly preserved region.

The "target sequence" which is not equal to any of the desaturase indicated (figure 2) , was then defined. The term

"target sequence" refers to the sequence on which helixes I and III which flank the catalytic domain of the ribozyme and which are complementary to the target sequence itself, are designed.

The regions flanking the cleavage triplet are not perfectly complementary with any of the target RNA and the Xs can be substituted by any of the four nucleotides. It is this particular characteristic which allows the ribozyme to exert its catalytic action on a larger number of RNA.

For illustrative purposes, a target sequence was defined, on which helixes I and III which flank the catalytic domain of the ribozyme Luna 9, were synthesized. More specifically, the differences between helix I of the ribozyme and the target gene sequence are: 0 for Linum usitatis- simum, 1 for Brassica napus and Olea europaea, 2 for He- lianthus annuus, Ricinus communis and Simmondsia chinensis, 3 for Carthamus tinctorius and Sesamum indicum.

The differences between helix III and the target gene sequence are: 1 for Linum usitatissimu and Brassica napus, 2 bases for Carthamus tinctorius, Helianthus annuus, Ricinus communis and Simmondsia chinensis and 3 bases for Olea europaea and Sesamum indicum.

On the basis of the sequence thus identified, the oli- gonucleotides complementary to helixes I and III were then synthesized, which, after pairing, were cloned in a vector for in vitro transcription. The cloning was effected following the standard proce- dures (Sambrook J. et al. (1989) Molecular cloning, A laboratory Manual, Cold Spring Harbor Laboratory Press, New York) . After each sub-cloning, the DNA encoding the ribozyme was sequenced to verify that there were no point muta- tions .

The transcribed ribozyme was used in catalytic tests (cleavage) in vitro to determine the cut efficiency on the stearoyl-acyl-carrier protein desaturase coming from various species (flax, rape, Castor oil plant, sunflower, olive) .

The sequence encoding the ribozyme can be put under the control of regulating regions which allows its expression in eukaryotic cells .

Examples of these regulating regions include promoters of the constitutive type or of viral origin (CaMV 35S, TMV) or so-called housekeeping genes (ubiquitina, actina, tubu- lina) associated with their termination sequence or with a heterologous one .

These regions also comprise specific tissue promoters, such as those of the genes involved in the biosynthesis of fatty acids (ACPs, acyltransferase, desaturase, lipid transfer protein genes) or those of the genes of seed reserve proteins (zein, napin, cruciferin, conglycin) associated with their termination sequence or with a different one . Inducible promoters associated with their own termination sequences, can also be used.

Finally, the plant expression cassette, described above, can be transferred to a vector which also contains a gene marker for the selection of transformed vegetable cells (for example genes for resistance to hygromycin, kanamycin, metotrexate, phosphinotrycin) .

The marker for the selection is under the control of a constitutive promoter. The vector is constructed so that both the transgene and the gene marker are both transferred into the plant genome.

The vegetable tissue used in the transformation can be obtained from any oleaginous plant .

These plants can be of the following kinds: Brassica, Helianthus, Carthamus, Sesamum, Glycine, Arachis, Gossyp- ium, Ricinus, Linum, Cuphea, Euphorbia, Limnanthes, Crambe,

Lesquerella, Vernonia, Simmondsia, Olea, Papaver, Elaeis,

Cocos and Zea.

Examples of suitable tissues for the transformation include leaves, hypocotyls, cotyledons, stems, calluses, single cells and protoplasts.

The transformation techniques which can be used are those well known in literature on the transformation of plants and include the transformation mediated by Agrobac- terium, electroporation, polyethylene glycol (PEG) and the use of the Particle Gun. All these systems allow insertion of the transgene and selection marker into the plant genome.

The transformed calluses can be selected by developing them on a selective substrate, such as, for example, a medium which contains the toxic chemical substance whose resistance gene has been transferred into the plant as gene marker.

Samples of tissues are removed from the regenerated plants, on which molecular analyses (Southern or PCR) are effected to determine the clones in which the transgene has been integrated in the genome .

The positive plants for the transgene can be seeded and the seeds are analyzed to determine the composition in fatty acids of the extracted oil . The characteristics of new fatty acids are determined by comparing the composition of transgenic genes with those of parental plants .

Genotypes whose seeds have shown an altered composition of fatty acids with respect to the wild type are mul- tiplied and subjected to a study of the stability of the nature and heredity through appropriate crossings. The characteristics conferred by the ribozyme can be transferred into agronomically valid varieties through traditional crossing. The following examples are provided for a better un- derstanding of the invention and should in no way be considered as limiting its scope. EXAMPLE 1

The sequences of the stearoyl-ACP desaturase examined are those of Linum usitatissimum, Brassica napus, Ricinus communis, Helianthus annuus, Carthamus tinctorius, Simmondsia chinensis, Olea europaea and Sesamum indicum.

The stearoyl-ACP Δ9 desaturase were compared using Clustal ^R multiple alignment software and a cleavage site (ATC) was identified in a highly preserved region.

The target sequence, which is not equal to any of the desaturase indicated, was then defined. On the basis of this sequence, the sequences of the two helixes (I and III) which flank the catalytic domain of the ribozyme and which are complementary to the target sequence (Figure 2) , were then determined.

More specifically, the differences between helix I of the ribozyme (SEQ. ID Nr. 2) and the sequence of the target gene are: 0 for Linum usitatissimum, 1 for Brassica napus and Olea europaea, 2 for Helianthus annuus, Ricinus communis and Simmondsia chinensis and 3 for Carthamus tinctorius and Sesamum indicum.

Helix III has the difference of 1 base for Linum usitatissimum and Brassica napus, 2 bases for Carthamus tinc- torius, Helianthus annuus, Ricinus communis and Simmondsia chinensis_. and 3 bases for Olea europaea and Sesamum indicum.

The length of the ribozyme, called Luna 9, is 48 bp

(SEQ. ID Nr. 2) to which the restriction sites BamHI are added, at both ends, for the subsequent cloning in Pgem3Zf

(Promega) . The length of the helixes I and III is 12 bases.

The two helixes of complementary DNA were synthesized as oligonucleotides by Roche Diagnostics SpA.

The two oligonucleotides (10 μg) were paired in 60 μl of buffer having the following composition: 10 mM TrisHCl pH 7.4, 100 mM NaCl, 0.25 mM EDTA, pH 8.

The solution was maintained at 68°C for 5 minutes and then left to cool slowly. The two oligonucleotides thus paired were precipitated with 2 volumes of ethanol at 100% and 1/10 of sodium acetate 3M pH 5.3, resuspended in 20 μl of water and 5 μg were subsequently digested with BamHI .

The digestion product, after being charged onto aga- rose gel at 2% was purified from gel with the GeneCleanTM kit (BIO 101 Inc, USA) . About 10 ng of DNA thus isolated were ligated to 50 ng of plasmid pGem3Zf (Promega) previously digested with the enzyme BamHI in 10 μl of reaction mixture, in the presence of 2 units of T4 DNA ligase, at 16°C for a night.

5 μl of this mixture were used to transform competent cells of E.coli DH5a (BRL) . The transforming agents were selected .on plates of LB medium (NaCl 10 g/1, Yeast extract 5 g/1, Bacto-triptone 10 g/1 and agar 20 g/1) containing 50

μg/ml of Ampicillin.

The plasmid DNA extracted from 5 positive clones was subjected to sequence analysis to verify the nucleotidic correspondence with the ribozyme indicated in Seq. ID Nr. 2.

The reactions and sequence analyses were carried out using the "Dye Terminator Cycle Sequencing Kit" according to the instructions of the producer PeBiosystems using the automatic sequencer "373 DNA Sequencer" of Applied Biosys- tems.

One of the clones analyzed, containing a fragment of DNA having SEQ: ID Nr. 2, was called MA399. EXAMPLE 2

Demonstration of the ribozyme activity in vitro

The ribozyme synthesized as described in Example 1, was reacted with the transcripts marked with radio-active phosphorous of some target genes (stearoyl-ACP desaturase of rape, flax, sunflower, olive and Castor oil plant) . The ribozyme was obtained by SP6 polymerase transcription of the SEQ ID Nr. 2 cloned in Smal linearized pGem3Zf .

The sense transcripts of the desaturase genes were obtained by linearizing the different plasmids in which they were cloned with an enzyme situated at 3 ' of the gene and at least .100 bp after the cleavage site (Table 1) .

Table 1: List of the Δ9 desaturase used in the in vitro tests, the enzymes used for the linearization, the polymer- ase used for the transcription. The last two columns indicate the dimensions of the whole transcript and those of the fragments generated by the catalytic action of the ribozyme.

Table 1

The Ambion Kit called MEGAscript™ was used for the transcription. The reaction mixture, prepared as described by the producer, consists of:

• 2 μl of reaction buffer;

• 2 μl of ATP (75mM for T7 and 50 mM for SP6) ;

• 2 μl of GTP 75 mM for T7 and 50 mM for SP6) ;

• 2 μl of CTP 75 mM for T7 and 50 mM for SP6) ;

• 2 μl of UTP 75 mM for T7 and 50 mM for SP6) ;

• 1 μl Of [α-32P] CTP ;

• 1 μg of linearized plasmidic DNA, • 2 μl of T7 polymerase or SP6 polymerase depending on the plasmid used and

• H₂0 so as to bring the volume to a total of 20 μl.

The mixture was incubated for 2 hours at 37°C and the plasmid DNA was subsequently degraded by adding 1 μl of KNase I Rnase-free and incubating for a further 15 minutes at 37°C.

The non-incorporated nucleotides were removed by means of precipitation with LiCl 2.5 M (final concentration). An estimate of the quantity of marked transcript obtained was effected by reading on the scintillator 1 μl of the transcription immediately before precipitation with LiCl (A) and 1 μl of the transcript resuspended in an equal volume after precipitation (B) . The ratio between B/A multiplied by 100 expresses the incorporation percentage of the marked nucleotide. The incorporation percentage multiplied by the maximum theoretical quantity of RNA which can be synthesized (calculated by assuming that all the nucleotides included in the mixture were used for the formation of RNA) expresses the quantity of transcript obtained.

When T7 polymerase was used, the maximum quantity of synthesizable RNA is 198 μg, in the case of SP6 polymerase, it is 132 μg. In the case of transcription with T7 polymerase, the concentration of each of the four ribonucleotides is equal to 7.5 mM (in the case of SP6 polymerase, it is equal to 5 mM) in a 20 μl reaction, this means that if all the nucleotides present in the mixture were used for the formation of RNA, the maximum quantity obtainable would be equal to 198

μg, i.e. to the product of:

(weight of 1 M of ribonucleotide x 4)/l000 mM x 7.5 mM/l06 x 20 μl = 198 μg i.e. by substituting: (330 x 4)g/l000 mM x 7.5 mM/l06 μl x 20μl = 198 μg.

10 nanograms of the transcript thus quantified, after denaturation at 65°C for 5 minutes, were incubated at 37°C for 15 minutes with an excess of the ribozyme transcript in a buffer having as final concentration 100 mM TrisHCl pH 7.6 and 50 mM MgCl₂. The cleavage reaction products were separated on polyacrylamide at 5%, 8 M Urea.

At the end of the electrophoretic run, the gel was dried and exposed using a phosphor screen of Molecular Dynamics which has the capacity of capturing the images com- ing from radio-active samples. The screen was then read using a Storm^R 860 scanner of Molecular Dynamics.

The cleavage percentage was estimated as the relative intensity of the autoradiographic signal determined by the use of Storm 860 and with the aid of Image-Quant software. By using the Phosphorimager^R and annexed software, it was possible to attribute a value directly correlated with the signal intensity to each band present on the gel . This value can be indicated as a percentage considering the sum of all the autoradiographic signals inside each line as equal to 100. As the bands inside each line can be attributed to the transcript of the target gel and its possible cleavage products, it is thus possible to express as a percentage the part of the target that has been cut .

On incubating the transcripts of rape, flax, sun- flower, Castor oil plant and olive with the ribozyme, an effective and highly specific cut was obtained, with the production, for each transcript, of two fragments having the expected dimensions .

More specifically in the case of stearoyl-ACP of flax, two fragments are obtained, deriving from the catalytic action of the ribozyme equal to 767 and 558 bases; in rape equal to 748 and 559 bases; in Castor oil plant 707 bases and 961 bases; in sunflower for Sad6 675 bases and 439 bases, whereas for Sadl7 681 bases and 614 bases; in olive 694 and 107 bases (Figures 3-4-5) .

The catalytic efficiency of the ribozyme obtained transcribing the sequence of the present invention is indicated in Tables 2 , 3 and 4 and was determined as described above. In the above tables, the line entitled "Line" refers to the path in which the marked gene transcript and the ribozyme transcript were charged contemporaneously. The lines in which the gene transcript alone was charged, were not taken into consideration, as in this case there was no cut. The fragments observed in each line are respectively distinguished by: T = entire residual transcript; 1 and 2 = products of the ribozyme catalytic action. The values of the column "intensity %" refer to the relative intensity of the signal of the fragments present in the lines; whereas the column "cleavage %" indicates the sum of the percentages relating to the intensity of the bands produced by the cleavage . The sum of the % referring to the peaks generated by the cleavage bands represents an estimate of the cut efficiency. Table 2. Catalytic efficiency of the ribozyme on the stearoyl-ACP desaturase of flax, rape, sunflower and Castor oil plant .

The data provided in this table refer to the test according to figure 3. This table does not indicate the olive value as the second cleavage fragment, of about 100 bases, was no longer visible in the gel. Table 2

Table 3. Cut efficiency on the stearoyl-ACP desaturase of sunflower, rape and flax.

The data provided in this table refer to the test according to figure 4. This table does not indicate the calculation relating to Sadl7 of sunflower (line 3) as the high residue could modify its determination.

Table 3

Table 4: Cut efficiency on the stearoyl-ACP desaturase of Castor oil plant, rape, flax and sunflower.

The data provided in this table refer to the test according to figure 5.

This table does not indicate the Olive value as the second cleavage fragment, of about 100 bases, was no longer visible in the gel. Table 4

From the data provided in the tables, it can be observed that, in spite of the imperfect complementarity of helixes I and III of the ribozyme with the gene target, the cleavage efficiency is equal to 100% for rape, flax and Castor oil plant, and, although reduced, it remains high in the two SAD of sunflowers . The cleavage tests were repeated several times and produced comparable results .

Claims

1. A Hammerhead ribozyme capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme of different oleaginous plants character- ized by SEQ. ID Nr.3.

2. A Hammerhead ribozyme according to claim 1, characterized by the sequence SEQ. ID Nr.4.

3. A DNA sequence which encodes the ribozyme of claim 1, characterized by SEQ. ID Nr.l.

4. A DNA sequence which encodes the ribozyme of claim 2, characterized by SEQ. ID Nr.2.

5. A vector selected from a plasmid or phage comprising the nucleotidic sequence SEQ.ID Nr.1 or 2.

6. A recombinant plant expression vector comprising the nucleotidic sequence SEQ. ID Nr.l or 2.

7. Prokaryotic or eukaryotic cells transformed with the vector of claim 6.

8. A method for the deactivation of the stearoyl-ACP desaturase enzyme of different oleaginous plants which comprises the reaction between the sequence which encodes the stearoyl-ACP desaturase enzyme and the ribozyme according to claim 1.

9. Transgenic plants comprising the nucleotidic sequence SEQ. ID Nr.l or SEQ. ID Nr.2 in their cells.

10. Seeds obtained from the transgenic plants according to claim 9