ITMI20011364A1 - ROBOZIMA HAMMERHEAD SPECIFIC FOR STEAROYL-ACP DESATURESI OF DIFFERENT OILAGINOUS PLANTS - Google Patents

Description

"Hammerhead ribozyme specific for the Stearoyl-ACP desaturase of different oil plants"

Description

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

In nature, lipids consist of saturated fatty acids, such as paimitic and stearic, and mono and polyunsaturated fatty acids, such as oleic, linoleic and linolenic. Their characterization is linked to the percentage with which each of these fatty acids is present.

Generally, solid lipids at room temperature are called fats and are characterized by a high percentage of saturated fatty acids.

Said acids are made up of linear molecules which tend to form a "stacked" structure responsible for their high melting point. The reduced presence of double bonds also gives these acids greater resistance to high temperatures and photooxidative processes. Animal fats are the main natural source of saturated fatty acids.

Lipids with a high percentage of mono- and poly-unsaturated fatty acids, on the other hand, are characterized by a lower melting point directly correlated with the degree of unsaturation of the fatty acids that compose them. They are liquid at room temperature and are commonly referred to as oils. The main natural source of mono- and polyunsaturated fatty acids is vegetable oils.

Both fats and oils are important for the processing industry, even if their intended use is different because it is linked to the different chemical-physical characteristics described above.

More precisely, the use of fats is particularly required in the confectionery industry and in all those food preparation processes that subject the fat to high temperatures.

Oils are mainly used in food and, those with a high content of polyunsaturated fatty acids, above all, in the industries of cosmetics, soaps, detergents, lubricants, paints, plastics, etc.

Over the years, animal-derived fats have been gradually replaced by vegetable oils as the latter do not contain cholesterol which is instead present in the membranes of animal cells.

Currently, oils of vegetable origin make up about 85% of the production of edible oils and fats and represent an essential element in human nutrition as they provide up to 25% of the caloric intake (Broun P. et al., 1999, Genetic engineering of plant lipids. Annu. Rev. Nutr. 19: 197-216).

Since these oils are rich in polyunsaturated fatty acids, it was necessary to subject them to a process of catalytic hydrogenation to reduce the double bonds.

This process allows to increase the melting point of the oil and to confer a better aroma and greater resistance to oxidative reactions, to which unsaturated fatty acids and particularly linolenic acid are susceptible.

The modified oil is used for the production of margarines which are often used as substitutes for butter in the confectionery and food industry.

However, the catalytic hydrogenation process, in addition to having an economic impact, causes the formation of trans-isomers of fatty acids which, when ingested, cause an increase in the level of cholesterol in the blood.

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

To overcome these drawbacks, new genetic engineering approaches have been proposed to modify the composition of vegetable oils in order to optimize them with respect to the needs of the human diet.

These systems are based on the partial or total inhibition of the expression of the genes that code for the desaturases involved in the metabolic pathway of fatty acids through the antisense technique (U.S. Pat. No.

5,850,026), co-oppression (Knutzon DS., Et al., Proc. Nati. Acad. Sci. USA, 1992, 89: 2624-28), or the use of multimeric ribozymes (Merlo A.O., et al., The Plant Cell., 1998, 10: 603-21).

As for the inhibition strategies mentioned above, the use of ribozymes has advantages due to their catalytic mechanism which requires few molecules to be effective.

In fact, the modulation of the expression of an endogenous gene, in the case of the antisense strategy or cosoppression, requires a stoichiometric interaction between the transgene and the target sequence.

This condition is no longer necessary if ribozymes are used, as a catalytic RNA is theoretically capable of cutting more molecules than the target RNA. Furthermore, the use of ribozymes as transgenes reduces the probability of the occurrence of suppression phenomena often associated with transformation with sense or antisense constructs. This is probably due to the small size of the transgene and the lower sequence homology with endogenous genes.

Ribozymes are circular RNAs able to catalyze their own fragmentation in the absence of proteins and in correspondence with particular nucleotide sequences.

In particular, the Hammerhead ribozymes (Haseloff et al., U.S. Patent N ° 6,127,114) represent a specific class of ribozymes and have the structure of the catalytic domain characterized by the presence of 3 helices (I, II, III) of variable length (Figure 1 ).

Such ribozymes consist of:

- a sequence of 22 nucleotides which constitutes the catalytic domain; And

- two regions flanking this catalytic domain which are complementary to the target RNA. These sequences allow the alignment of the target RNA with the catalytic domain and confer specificity to the reaction.

All the ribozymes so far designed are characterized by having a high specificity towards the target RNA. This specificity is conferred to the ribozyme by the perfect complementarity of the two helices flanking the catalytic domain with the target RNA.

It has now been found that, by using a single, appropriately designed Hammerhead-type ribozyme, it is possible to modulate the expression of the gene encoding the stearoyl-ACP desaturase enzyme of different oil plants.

Accordingly, an object of the present invention is a Hammerhead ribozyme capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme of different oil plants characterized by the fact that the two regions flanking the catalytic site are not perfectly complementary. with none of the target sequences.

A further object of the present invention is a DNA sequence which codes for said ribozyme.

An object of the present invention also constitutes an expression vector in plants containing the DNA sequence which codes for said ribozyme.

Another object of the present invention is a method for increasing the stearic acid content in different oil plants which uses said ribozyme.

Furthermore, an aim of the present invention is transgenic plants transformed with said ribozyme.

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

Brief description of the figures

Figure 1: the secondary structure of a Hammerhead-type ribozyme is reported.

The central box contains the catalytic domain of the ribozyme (helix II); the two lateral boxes constitute, proceeding from left to right, the helix III and the helix I of the ribozyme. The Xs indicate the nucleotides of the target sequence that pair up with the two helices 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 drawn (see Figure 2). This nucleotide base associated with U and C constitutes the cleavage triplet indicated in bold.

Figure 2: A comparison is reported between the target sequence defined in the present invention and the target sequences of some oil plants.

The central ATCs in bold and underlined represent the cleavage triplet; the bases in bold, indicated in the sequences of the different species, are the bases different from the target sequence.

Figure 3:

The photo of the gel relating to the catalytic assay is shown on the transcript of the stearoyl-ACP desaturase of some species.

Line 1: Linen; Line 2: Linen ribozyme; Line 3: Rapeseed; Line 4: Rapeseed ribozyme; Line 5: Sad 6 (Girasole); Line 6: Sad 6 (Girasole); ribozyme; Line 7: Sad 17 (Girasole); Line 8: Sad 17 (Girasole) ribozyme; Line 9: Olivo; Line 10: Olivo ribozyme; Line 11: Castor; Line 12: Castor ribozyme.

In Lines 1, 3, 5, 7, 9 and 11 a single band appears which is that relating to the labeled transcript of the gene; while in Lines 2, 4, 6, 8, 10 and 12 the two bands produced by the cut of the target gene by the ribozyme are evident.

Figure 4:

The photo of the gel relating to the catalytic assay is shown on the transcript of the stearoyl-ACP desaturase of some species.

Line 1: Sad 17 (Girasole) ribozyme; Line 2: Sad 17 (Sunflower); Line 3: Sad 6 (Sunflower) ribozyme; Line 4: Sad 6 (Girasole); Line 5: Rapeseed ribozyme; Line 6: Rapeseed; Line 7: Linen ribozyme; Line 8: Linen.

Figure 5:

The photo of the gel relating to the catalytic assay is shown on the transcript of the stearoyl-ACP desaturase of some species.

Line 1: Olivo ribozyme; Line 2: Olivo; Line 3: Castor ribozyme; Line 4: Castor; Line 5: Rapeseed ribozyme; Line 6: Rapeseed; Line 7: Linen ribozyme; Line 8: Linen; Line 9: Sad 17 (Girasole) ribozyme; Line 10: Sad 17 (Girasole);

Detailed description of the invention

In the seeds of oil plants, the synthesis of fatty acids appears to occur in the plastids and subsequently these acids are transported to the endoplasmic reticulum, where they are used for the formation of triglycerides.

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

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

In order to construct a ribozyme capable of interacting with a wider range of target RNAs having a region of high homology between them, the sequences of the Δ9 desaturases of Linum usitatissimum, Brassica napus, Ricinus communis, Helianthus annuus were considered. , Carthamus tinctorius, Simmondsia chinensis, Olea europaea and Sesamum indicum.

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

We then proceeded to define the "target sequence" which is not equal to any of the desaturases reported (Figure 2). The term "target sequence" defines the sequence on which helices I and III flanking the catalytic domain of the ribozyme and which are complementary to the target sequence itself are drawn.

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

For example, a target sequence has been defined on which helices I and III flanking the catalytic domain of the Luna 9 ribozyme have been synthesized. More precisely, the differences between the helix I of the ribozyme 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, 3 for Carthamus tinctorius and Sesamum indicum.

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

Then, on the basis of the sequence thus identified, the oligonucleotides complementary to helices I and III were synthesized which, after pairing, were cloned into a vector for in vitro transcription.

Cloning was performed following standard procedures (Sambrook J. et al. (1989) Molecular cloning, A laboratory Manual, Cold Spring Harbor Laboratory Press, New York). After each subcloning, the DNA encoding the ribozyme was sequenced to verify that there were no point mutations.

The transcribed ribozyme was used in catalytic assays ("cleavage") in vitro to determine the shear efficiency on stearoyl-acyl-carrier protein desaturase from various species (flax, rapeseed, castor, sunflower, olive).

The sequence encoding the ribozyme can be placed under the control of regulatory regions that allow its expression in eukaryotic cells.

Examples of such regulatory regions include promoters of constitutive type or of viral origin (CaMV 35S, TMV) or the so-called "housekeeping genes" (ubiquitin, actin, tubulin) associated with their termination sequence or with a heterologous one.

These regions also include 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 seed reserve protein genes (zein, napin, cruciferin , conglicina) associated with its own termination sequence or with a different one.

In addition, inducible promoters associated with their own termination sequences can be used.

Finally, the plant expression cassette, described above, can be transferred to a vector that also contains a marker gene for the selection of transformed plant cells (e.g. genes for resistance to hygromycin, kanamycin, methotrexate, phosphinothricin).

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

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

Such plants can be, for example, of the genus Brassica, Helianthus, Carthamus, Sesamum, Glycine, Arachis, Gossypium, Ricinus, Linum, Cuphea, Euphorbia, Limnanthes, Crambe, Lesquerella, Vernonia, Simmondsia, Olea, Papaver, Elaeis, Cocos and Zea .

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

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

The transformed calluses can be selected by having them develop on a selective substrate, such as a medium that contains the toxic chemical whose resistance gene has been transferred to the plant as a marker gene.

Tissue samples are taken from regenerated plants for molecular analyzes (Southern or PCR) to determine the clones in which the transgene has integrated into the genome.

Plants positive for the transgene can be brought to seed and the seeds are analyzed for the determination of the fatty acid composition of the extracted oil. The characteristics of new fatty acids are determined by comparing the composition of the transgenic seeds with those of the parent plants.

The genotypes whose seeds showed an altered composition of fatty acids compared to the wild type are multiplied and subjected to the study of the stability of the character and heredity through appropriate crosses. The characteristics conferred by ribozyme can be transferred into agronomically valid varieties through traditional crossbreeding.

The following examples are given for a better understanding of the invention and should not be considered as limiting thereof.

Example 1

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

With ClustalWR multiple alignment software, stearoyl-ACP Δ9 desaturases were compared and a cleavage site (ATC) was identified in a highly conserved region.

We then proceeded to define the target sequence which is not equal to any of the desaturases reported. On the basis of this sequence, the sequences of the two helices (I and III) which flank the catalytic domain of the ribozyme and which are complementary to the target sequence were then determined (Figure 2).

More precisely, the differences between helix I of ribozyme (SEQ.ID N ° 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 presents the difference of 1 base for Linum usitatissimum and Brassica napus, 2 bases for Carthamus tinctorius, Helianthus annuus, Ricinus communis and Simmondsia chinensis and 3 bases for Olea europaea and Sesamum indicum.

The length of the ribozyme, called Luna9, is 48 bp (SEQ.ID N ° 2) to which the BamHI restriction sites must be added at both ends for subsequent cloning into pGEM3Zf (Promega). The length of helixes I and III is 12 bases.

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

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

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

The digestion product, after being loaded on 2% agarose gel, was purified from gel with the GeneCleanTM kit (BIO 101 Ine, USA).

About 10 ng of the DNA thus isolated were ligated to 50 ng of plasmid pGem3Zf (Promega) previously digested with the BamHI enzyme in 10 μl of the reaction mixture, in the presence of 2 units of T4 DNA ligase, at 16 ° C for a night.

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 μg / ml of ampicillin.

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

Reactions and sequence analyzes were conducted using the "Dye Terminator Cycle Sequencing Kit" according to manufacturer PEBiosystems' instructions using the Applied Biosystems "373 DNA Sequencer automatic sequencer.

One of the analyzed clones, containing a DNA fragment having the SEQ: ID No 2, was named MA399.

Example 2

Demonstration of ribozyme activity in vitro.

The ribozyme synthesized as in Example 1 was reacted with the radioactive phosphorus-labeled transcripts of some target genes (stearoyl-ACP desaturase from rapeseed, flax, sunflower, olive and castor). The ribozyme was obtained by transcribing with the SP6 polymerase the SEQ ID No: 2 cloned in pGem3Zf linearized with the Smal enzyme.

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

Table 1: List of Δ9 desaturases used in in vitro assays, enzymes used for linearization, polymerases used for transcription. The last two columns show the dimensions of the intact 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 manufacturer, consists of: • 2 μl of reaction buffer;

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

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

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

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

• 1 μl of [a-32P] CTP;

• 1 μg of linearized plasmid DNA,

• 2 μl of T7 polymerase or SP6 polymerase depending on the plasmid used e

• H2O in order to bring the volume to 20 μl total;

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

The unincorporated nucleotides were removed by precipitation with Liei 2.5 M (final concentration). An estimate of the quantity of labeled transcript obtained was performed by reading at the scintillatone 1 μl of the transcription mixture 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 percentage of incorporation of the labeled nucleotide. The percentage of incorporation multiplied by the theoretical maximum quantity of synthesizable RNA (calculated assuming that all the nucleotides included in the mixture have been used for the formation of RNA) expresses the quantity of transcript obtained.

In the case in which T7 polymerase has been 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 reaction of 20 μl, 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. the product of:

(weight of 1 M ribonucleotide x 4) / 1000 mM x 7.5 mM / 106 x 20 μl = 198 μg

that is, substituting:

(330x4) g / 1000 mM x 7.5m M / 106 μl x 20ul = 198 μg

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

At the end of the electrophoretic run, the gel was dried and exposed using the Molecular Dynamics "phosphor screen" which has the ability to capture images from radioactive samples. The screen was then read using Molecular Dynamics' Storm <R> 860 scanner.

The percentage of cleavage was estimated as the relative intensity of the autoradiographic signal determined by the use of the Storm 860 and the aid of the Image-Quant software.

Using the Phosphorimager <R> and the related software it was possible to attribute to each band present on the gel a value directly correlated with the intensity of the signal. This value can be indicated as a percentage considering the sum of all autoradiographic signals within each line to be equal to 100. Since the bands within each line are to be attributed to the presence of the transcript of the target gene and its possible cleavage products, it is possible in this way to express in percentage how much of the target has been cut.

By incubating the transcripts of rapeseed, flax, sunflower, castor and olive with ribozyme, an efficient and highly specific cut was obtained with the production, for each transcript, of two fragments having the expected dimensions.

More precisely, in the case of linen stearoyl-ACP, two fragments deriving from the catalytic action of the ribozyme equal to 767 and 558 bases are obtained; in rapeseed equal to 748 and 559 bases; in castor oil 707 bases and 961 bases; in sunflower for the Sad6 675 bases and 439 bases, while for the Sadl7681 bases and 614 bases; in the olive tree 694 and 107 bases (Figures 3-4-5).

The catalytic efficiency of the ribozyme obtained by transcribing the sequence of the present invention is reported in Tables 2, 3 and 4 and was determined as described previously.

In the above tables the row called "Line" refers to the run in which the labeled transcript of the gene and the transcript of the ribozyme have been loaded at the same time. The lines in which only the transcript of the gene was loaded were not considered, as in this case there was no cut.

The fragments that are highlighted in each line are marked respectively by: T = intact residual transcript; 1 and 2 = products of the catalytic action of the ribozyme. The values in the "% intensity" column refer to the relative intensity of the signal of the fragments present in the lines; while the "% of cleavage" column shows the sum of the percentages relating to the intensity of the bands produced by the cleavage. The sum of the% referred to the peaks generated by the cleavage bands represents an estimate of the cutting efficiency.

Table 2. Catalytic efficiency of ribozyme on stearoyl-ACP desaturase of flax, rapeseed, sunflower and castor.

The processing reported in this table refers to the assay in figure 3. This table does not report the calculation relating to the olive tree as the second fragment of cleavage, of about 100 bases, was no longer visible in the gel.

Table 2

Table 3. Cutting efficiency on the stearoyl-ACP desaturase of sunflower, rapeseed and flax.

The processing reported in this table refers to the rate in figure 4. This table does not report the calculation relating to the sunflower Sad17 (line 3) as the high background could alter its determination.

Table 3.

Table 4: Shear efficiency on castor, rapeseed, flax and sunflower stearoyl-ACP desaturases.

The processing reported in this table refers to the assay shown in Figure 5.

This table does not show the calculation relating to the Olive tree as the second fragment of cleavage, of about 100 bases, was no longer visible in the gel.

Table 4.

From the data reported in the tables it is observed that, despite the imperfect complementarity of helices I and III of the ribozyme with the target gene, the cleavage efficiency is equal to 100% for rapeseed, flax and castor oil, and, even if it is reduced, it remains high. in the two SADs of the sunflower. The cleavage tests were repeated several times and produced comparable results.

Claims (9)

Claims 1. A Hanunerhead ribozyme capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme of different oil plants characterized by SEQ.ID N ° 3. 2. A Hammerhead ribozyme according to claim 1, characterized by the sequence SEQ.ID N ° 4. 3. A DNA sequence encoding the ribozyme of claim 1, characterized by SEQ ID No. 1. 4. A DNA sequence encoding the ribozyme of claim 2, characterized by SEQ ID No. 2. 5. A vector selected from a plasmid or phage comprising the nucleotide sequence SEQ ID No. 1 or 2. 6. A recombinant vector for plant expression comprising the nucleotide sequence SEQ ID No. 1 or 2. 7. Prokaryotic or eukaryotic cells transformed with the vector of claim 6. 8. A method for the inactivation of the stearoyl-ACP desaturase enzyme of different oil plants which comprises the reaction between the sequence encoding the stearoyl-ACP desaturase and the ribozyme of claim 1. 9. Transgenic plants including the nucleotide sequence SEQ ID No: 1 or SEQ ID No: 2 in their cells. 0. Seeds obtained from transgenic plants of claim 9.