NZ235181A - Rna with ribonuclease activity for mrna of ripening genes - Google Patents

Abstract

Ribozyme genes or ribozyme gene fragments can be synthesised on the basis of the c-DNA of ripening genes. They are then introduced into plant cells where they are expressed, causing virtually complete inhibition of the ripening enzymes.

Description

New Zealand Paient Spedficaiion for Paient Number £35181 235 181 Priority MK'J- .... ; Publication Oai; "0 y.y-V'1*! i1- *_§ 11 HO DRAWINGS = .5SEPW°"j N.2. No.

NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION RNA WITH ENDORIBONUCLEASE ACTIVITY FOR MRNA OF RIPENING GENES. THE PREPARATION THEREOF AND THE USE THEREOF IN PLANTS We, HOECHST AKTIENGESELLSCHAFT, a Joint Stock Company existing under the laws of the Federal Republic of Germany, of D-6230 Frankfurt am Main 80, Federal Republic of Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- - 1 - (Followed by 1A) Descxipt, ioii - 1 A - RNA with endoribonuclease activity for mRNA of ripening genes, the preparation thereof and the use thereof in 5 plants Under certain conditions, RNA molecules are able, without involvement of proteins, to catalyze reactions on other RNA molecules or to eliminate autocatalytically fragments from their own molecules. Thus, there is autocatalytic 10 deletion of an intron containing 413 nucleotides from the 3' end of the 23s rRNA of Tetrahymena thermophila, and conversion into a circular form. This takes place by a series of phosphoester transfer reactions in which guanosine cofactors are involved (Cech, T.R., Nature 30. 15 578-583 (1983)). Depending on the RNA substrate or the chosen reaction conditions, the intron can function as specific ribonuclease, terminal transferase, phosphotransferase or acid phosphatase. Moreover, one RNA molecule is able to carry out a number of conversions 20 without itself being changed and, in this respect, behaves like an enzyme. This is why the term ribozyme has been coined for RNA molecules with these properties.

Similar reactions without involvement of proteins have also been demonstrated for some viroidal RNAs and satel-25 lite RNAs. Thus, self-processing appears to be an essential reaction for replication of avocado sunblotch viroid (ASBV) (Hutchins, C.J. et al. Nucleic Acids Res. 14, 3627-3640 (1986)), satellite RNA of tobacco ringspot virus (sTobRV) (Prody, G.A. et al., Science 231. 1577-30 1580 (1986)) and satellite rna ox lucerne transient streak virus (sLTSV) (Forster A.C. et al.. Cell 49., 211-220 (1987)). It is supposed that circular forms are produced during the replication of these RNAs and lead, as templates, to the synthesis of RNAs with extensions. 35 These transcripts are cut to the correct genome length by the self-catalyzed endonucleolytic reactions. rv 1*' ^ d i fc The structures of the RNAs which presumably take the latter in for the reaction have been described as hammerheads (Forster, A.C. et al.. Cell 49., 211-220 (1987); Haseloff, J. et al., Nature 334, 585-591 (1988)).

The cleavage sites for these RNA enzymes are specific and must display particular structural requirements for processing to be able to occur.

It has now been found that ribozymes are able to attack plant RNA coding for ripening enzymes and thus can be 10 used for influencing the ripening processes in plants.

Regulation of the expression of the DNA coding for the ripening enzyme polygalacturonase by antisense RNA has been described by Smith C.J.S. et al. in Nature 334, 724 (1988). A fragment of the polygalacturonase cDNA is 15 placed in the opposite orientation in an expression vector. This vector plasmid is used to transform, via E. coli and Agrobacterium tumefaciens, stalk segments of the tomato. Expression of antisense RNA can then be detected in the leaves of the tomato plant. It is assumed that the 20 antisense RNA attaches itself to the actual polygalac turonase RNA, leading to inactivation of the latter, with the further consequence that there is partial inhibition of polygalacturonase synthesis.

Ribozymes which bind to ripening enzyme RNA and are able 25 to cleave the latter at particular cleavage sites in the sequence have now been developed for specifically influencing the ripening process in plants. It is possible with the aid of the ribozymes according to the invention for the synthesis of particular ripening 30 enzymes not just to be partially inhibited but to be virtually completely inhibited, i.e. about 80-100%. 1 .1. v <.<* ' Hence the invention relates to: 1. Genes or gene fragments encoding ribozymes, and the corresponding ribozyme RNA sequences and a process for the preparation thereof. 2. Plants, plant cells and parts or seeds of the plants which contain the DNA or RNA sequence specified under 1. 3. The use of ribozymes for inhibiting the synthesis of ripening enzymes in plants.

The invention is described in detail hereinafter, especially in its preferred embodiments. The invention is also defined in the claims.

The ribozyme can be synthesized on the basis of the DNA sequence of the ripening gene to be inhibited. In this 15 connection, it is possible in principle to start from any DNA sequence coding for a plant ripening enzyme. Examples of such plant ripening enzymes are polygalacturonase, pectin esterase and so-called ripening-related proteins. It is preferable to choose as "starter" for the synthesis 20 at least 10 consecutive nucleotides, in particular 14 to 20 nucleotides, advantageously from the middle of the cDNA sequence of the structural gene. It is particularly advantageous to start from the cDNA sequence for polygalacturonase (Grierson, D. et al., NAR 14. 8595 (1986)), 25 pectin esterase (Ray, J. et al., Eur. J. Biochem. 174, 119 (1988)) and for ripening-related protein (Ray, J. et al., NAR .15, 10587 (1987)) in each instance.

Chemical synthesis of oligonucleotides on the basis of the cDNA sequence is carried out in such a way that the 30 initial and final sequences of the oligonucleotides are each composed of 5, preferably 7 to 10, nucleotides which, taken together, are complementary to a DNA sequence of the ripening enzyme to be inhibited and that 23 5 1 the initial and final sequences of the oligonucleotides are separated by an interpolated RNA sequence which is composed partly of specific nucleotides predetermined for the functionality of the ribozyme and partly of variable 5 nucleotides. The appearance of the ribozyme hybridized with substrate RNA can be outlined as follows.

-NNNNNNNNNNNNNGUC NNNNNNNNNNNNNN-3 3-KKKKKKKKCA KKKKKKKK-5 A C A U G A CG G, V V V V ] substrate RNA G A 'V Loop ribozyme where N are nucleotides of the substrate RNA, A, C, G or T, K are complementary nucleotides to N in the ribozyme, V are variable nucleotides in the ribozyme and VL are variable nucleotides in the loop of the ribozyme.

The number of VL nucleotides in the loop can be 0-550. A GU recognition sequence is preferably chosen as cleavage site in the substrate RNA.

The said oligonucleotides are provided with an appropriate linker. Linkers of this type possess, for example, 20 cleavage sites for EcoRI, Sail, BamHI, Hindlll, EcoRV, Smal, Xhol, Kpnl, preferably Xbal and Pstl.

The synthesized oligonucleotides are cloned with the aid of the vectors pUCl9, pUC18 or pBluescript (Stratagene, Heidelberg, Product Information), and sequenced. The 25 verified oligonucleotide is cloned into an intermediary vector with a plant promoter. Examples of vectors of this type are the plasmids pPCV701 (Velten, J. et al. EMBO J. *> J V.

«. -J r- 3, 2723-2730 (1984)), pNCN (Fromm M. et al., PNAS 82, 5824-5826 (1985)) or pNOS (An G. et al., EMBO J. 4, 277-276 (1985)). The vector pDHSl (Pietrzak, M. et al.. Nucleic Acids Res. .14, 5857, (1986)) with a 35S promoter 5 is preferably used.

After subsequent transformation of E. coli, such as, for example, E. coli MC 1061, DH1, DK1, GM48 or XL-1, positive clones are identified by methods known per se (Maniatis et al., Lab. Manual), such as plasmid mini-10 preparation and cleavage with an appropriate restriction enzyme.

These positive clones are then subcloned into a binary plant vector. Plant vectors which can be employed are PGV3850 (Zambrysk, P. et al., EMBO J. 2, 2143-2150 15 (1983)) or pOCA18 (Olszewski, N., Nucleic Acids Res. 16., 10765-10782, (1988)). pOCA18 is preferably used.

The resulting binary plant vectors which contain a plant promoter with the attached DNA fragment for ribozyme production in the T-DNA are used to transform plants. 20 Techniques such as electroporation or microinjection can be employed for this.

Preferably employed is cocultivation of protoplasts or transformation of small pieces of leaf with Agrobacteria. For this, the plant vector construct is transferred by 25 transformation with purified DNA or, mediated by a helper strain such as E. coli SM10 (Simon R. et al.. Biotechnology .1, 784-791 (1983)), into Agrobacterium tumefaciens such as A282 with a ™i plasmid via triparen-tal meting. Direct transformation and triparental mating 30 were carried out as described in "Plant Molecular Biology Manual" (Kluwer Academic Publishers, Dordrecht (1988)).

It is possible in principle to transform all plants with binary plant vectors carrying ribozyme DNA. Dicotyledonous plants are preferred, especially productive plants such as, for example, fruit-bearing plants. Tomato, strawberry, avocado and plants which bear tropical fruits, for example papaya, mango, but also pear, apple, nectarine, apricot or peach, may be mentioned as ex-5 amples. The described process is particularly preferably carried out with the tomato. The transformed cells are selected with the aid of a selection medium, cultured to a callus and regenerated to the plant on an appropriate medium (Shain et al., Theor. appl. Genet. 72, 770-770 10 (1986); Masson, J. et al., Plant Science J53, 167-176 (1987), Zhan et al., Plant Mol. Biol. 11, 551-559 (1988); McGranaham et al., Bio/Technology 6, 800-804 (1988); Novrate et al., Bio/Technol. 2/ 154-159 (1989)).

The resulting plant is altered by the transformation in 15 such a way that the ribozymes are expressed in the cells, which in turn has the effect that the ribozyme RNA not only binds to the RNA complementary to the appropriate ripening genes and brings about more or less extensive inhibition of synthesis of the ripening enzyme, but that 20 the RNA complementary to the appropriate ripening genes is specifically cut at GUC sequences, which leads to almost complete inhibition of synthesis of the relevant ripening enzyme.

The formation of the ribozyme-specific secondary struc-25 tural features of the ribozyme RNA synthesized in the transgenic plant in vivo was entirely unexpected, so that the observed inhibition of synthesis of the ripening enzymes was completely surprising.

The examples which follow serve to illustrate the inven-30 tion further. *■"'5 181 Examples Unless indicated otherwise, percentage data relate to weight. 1. Cloning of the oligonucleotides The synthesis of the oligonucleotides for ribozyme expression was based on the cDNA sequence a) 5' TGATGGAGTCCATGTATCA 3' Section of the polygalact. cDNA sequence according to Grierson, D. et al., 10 Nucleic Acids Res. 14., 8595-8603 b) 5' TAGCAAGTCCTGACCTAA 3' Section of the cDNA sequence of pectin esterase according to Ray, J., Eur. 15 J. Biochem. 174, 119-124 (1988) c) 5' TGCTTTGTCCGATACAGT 3' Section of the cDNA sequence of a ripening-related protein according to Ray, J., Nucleic Acids Res. 15, 10587 (1987) The phosphoramidite method (Engels J. et al., Advances in Biochemical Engineering Biotechnology Volume 37 ed.: A. Fiechter, Springer Verlag, Berlin/Heidelberg, 1988) was 25 used to synthesize the following oligonucleotides in a synthesizer: for a) 5' CTAGATGATACATGCTGATGAGTCCGTGAGGACGAAACTCCATCTGCA 3' 3' TACTATGTACGACTACTCAGGCACTCCTGCTTTGAGGTAG 5' for b) 5' - CTAGATTAGGTCAGCTGATGAGTCCGTGAGGACGAAACTTGCTACTGCA- 3' 30 3' - TAATCCAGTCGACTACTCAGGCACTCCTGCTTTGAACGATG - 5' - 8 for C) 5' -CTAGACTGTATCGCTGATGAGTCCGTGAGGACGAAACAAAGCACTGCA-3' 3' -TGACATAGCGACTACTCAGGCACTCCTGCTTTGTTTCGTG- 5' The vector pDH51 (Pietrzak, H. et al.. Nucleic Acids Res. 14, 5857 19) was cut with the restriction endonucleases 5 Xbal and PstI, incubated with calf intestinal phosphatase (CIP), phenol-treated and precipitated (Maniatis, Lab. Manual). The vector treated in this way was ligated with a three-fold excess of phosphorylated oligonucleotides and transformed into E. coli MC1061. Positive clones were 10 identified by plasmid minipreparations and subsequent digestion with Xbal and PstI.

In addition, the ampicillin-resistant transformed E. coli cells (100 p.g of ampicillin per ml of LB medium) were transferred to nitrocellulose membranes (Gene Screen 15 Plus®, NEN®, Boston) and incubated on LB medium containing ampicillin at 37°C for a further 14 hours. The colonies were then disrupted in 0.5 M NaOH and fixed. After drying, it was possible to hybridize the filters with radiolabeled oligonucleotides. Positive clones produced 20 blackening on the film. 2. Subcloning of a 35S promoter gene fragment in pOCA 18 A 0.75 kb EcoRI fragment was isolated from each of the clones obtained as in 1. This fragment was inserted into a pOCA 18 vector which had been cut with EcoRI, and was 25 transformed into E. coli MC1061. Positive clones were identified by plasmid minipreparations and, after subsequent hydrolysis with EcoRI, by the appearance of the 0.75 kb band. 3. Transformation of Agrobacterium tumefaciens In order to be able to transform plants, the construct obtained as in 2. must be transferred into an Agrobacterium. This takes place either by triparental mating or directly. In the case of triparental mating, 100 pi • • T R samples of bacteria from overnight cultures of E. coli SM10, the E. coli MCI061 carrying the construct, and Agrobacterium tumefaciens were spun down and taken up together in 30 /*1 of LB medium. After 30 minutes at room 5 temperature, this bacteria suspension was placed on a filter on an LB plate without antibiotics. The filter was incubated at 37cC for 12 h and then washed in 2.5 ml of 10 mM MgCl2. Aliquots were selected on LB plates containing rifampicin, tetracycline and kanamycin. Positive 10 colonies were identified by hybridization with 32P-labeled DNA of the gene to be expressed.

In the case of direct transformation of Agrobacteria, the cells were cultured at 28°C overnight in YEB medium (1% yeast extract, 1% peptone, 0.5% NaCl) containing 25 nq/ml 15 kanamycin and 100 ^g/ml rifampicin. After 16 hours, the bacteria suspension was diluted to an ODS50 of 0.1 and incubated further at 28°C until the OD550 was 0.5. 1 ml of this culture was spun down and washed with 1 ml of 150 mM NaCl. After washing, the precipitate was resuspended in 20 600 ^1 of ice-cold 10 mM CaCl2 solution.

The pOCA 18 vector with the cloned 0.75 kb fragment was isolated from the E. coli clones obtained as in 2. after disruption of the cells with 0.2 N NaOH/1% SDS, was purified by CsCl density gradient centrifugation. 1 pg of 25 the plasmid DNA was added to the competent Agrobacteria, and the Eppendorf tubes were placed on ice for 1 hour. After 1 hour, the plasmid solution was incubated at 37 °C for 5 minutes, and 2 ml of YEB medium were added. The cells were then incubated at 28*C in a shaking incubator 30 overnight. Thereafter 100 pi samples were placed on YEB plates containing 100 nq/ml rifampicin, 25 /xg/ml kanamycin and 2.5 ^g/ml tetracycline. Colonies were found on the plates after 2 days at 28eC. Positive clones were detected by hybridization with the appropriate 32P-labeled 35 oligonucleotides a), b) or c).

For this, the transformed Agrobacteria were streaked on 3 5181 Gene Screen Plus membranes and incubated on YEB plates containing 100 rifampicin, 25 itq/ml kanamycin and 2.5 pg/ml tetracycline at 28°C for 14 hours. The membranes were then placed on 0.5 M NaOH for 2 minutes and 5 subsequently on 0.5 M Tris, pH 7.5, for 2 minutes. After drying, prehybridization was carried out in 10% dextran sulfate/1 M NaCl/1% SDS at 55cC for 2 hours, and then the radiolabeled oligonucleotides a), b) or c) were added. The filters were incubated together with the radiolabeled 10 oligos at 55°C overnight. After washing at 55°C for 30 minutes each with lx SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and 2x with 0.2 x SSC, the positive clones were identified by blackening of a film placed on top. 4. Transformation of tomatoes a) Protoplast transformation: Tomato protoplasts (Plant Cell Reports 6., 172-175 (1987)) are washed once with W5 solution (154 mM NaCl, 125 mM CaCl2 . 2 2 H20, 5 mM KC1, 5 mM glucose) and once with MaMg solution (0.45 M mannitol, 25 mM MgCl2, 0.1% 2-(N-morpholino)ethanesulfonic acid (MES, pH5.8) from Sigma Chemie, Deisenhofen, FR Germany). After careful centrifugation at 600 rpm for 3 minutes, the supernatant, apart from 0.5 ml, is aspirated off. To this are added by pipette 50 nq of calf thymus DNA, 10 nq of the described plasmid and 10 drops of 45% polyethylene glycol (PEG 8000). After 10 minutes, the protoplasts are washed twice with W5 solution and incubated in LCM medium (Plant Cell Reports 6., 172-175 (1987)). b) Transformation of small pieces of leaves with Agrobacteria: Tomato leaves are cut up small and placed on MS medium (Murashige, T. et al., Physiol. Plant 15, 473-497 35 (1962)) containing 2% sucrose and 1 ppm plant growth hormone zeatin (Serva Feinbiochemica GmbH & Co., 9U1 V- V.i 'V £ Heidelberg, FR Germany) and incubated at 25°C over a period of 16 hours light/day. Infection with Agrobacteria is carried out one day later. This entails the small pieces of leaves being briefly immersed in a 5 dilute bacteria suspension (OD 0.15) of the trans formed Agrobacterium strain, replaced on the same plate and further incubated under the same conditions. On the third day, all the small pieces of leaves are washed with 250 mg/1 carbenicillin solution and placed 10 on a 2MS medium containing 1 ppm zeatin, 200 mg/1 cefotaxime (Hoechst AG, Frankfurt), 200 mg/1 carbenicillin and 100 mg/1 kanamycin. Regenerants were transferred after approximately 20 more days to 2MS medium containing 1 ppm zeatin, 200 mg/1 cefotaxime, 15 200 mg/1 carbenicillin and 100 mg/1 kanamycin.

Detection of the ribozyme RNA of a transgenic tomato plant which is directed against the ripening-related protein RNA.

The total cellular RNA was isolated from a small piece of 20 leaf from a transgenic tomato plant which had been transformed with the ribozyme-encoding gene directed against the ripening-related protein RNA. For this, the leaf material was ground with a mortar and pestle under liquid nitrogen. The powdered leaf material was mixed 25 with twice the volume of an extraction buffer (0.2 M sodium acetate, 1% SDS, 10 mM EDTA), twice the volume of phenol (equilibrated with extraction buffer) and half the volume of chloroform/isoamyl alcohol 24:1 (v/v). After very thorough mixing, the phases were separated by 30 centrifugation and the phenolic extraction of the upper acrueous phase was repeated. This aqueous phase was, after mixing and separation by centrifugation, again transferred into a fresh Eppendorf tube and extracted with chloroform/ isoamyl alcohol 24:1 (v/v). Then 1/3 volume 35 of 8 M LiCl was added to the aqueous phase, and the mixture was maintained at 4#C overnight. A precipitate formed after centrifugation. This was taken up in water '23 5 1 and, after addition of 0.3 M sodium acetate (pH 5.5) (final concentration), washed with 2 1/2 volumes of ethanol. After washing with 70% ethanol and drying, the precipitate was taken up in 50 pi of water.

To detect the specific expression of the ribozyme-encod-ing gene, samples of about 4 f.ig of total RNA from a non-transformed wild-type tomato plant and from a transgenic tomato plant were applied to a 1% agarose gel with 6% formaldehyde. For the application, the RNA was dried, 10 taken up in 50% formamide/6% formaldehyde and heated at 60eC for 15 minutes. The agarose gel was briefly washed with H20 after the run and transferred with 10 x SSC to a Gene Screen Plus membrane. After 24 hours, the membrane was washed with 2 x SSC, incubated at 80eC for 2 h and 15 dried.

Hybridization was carried out after 2 hours' prehybridi-zation with 1% SDS, 1 M NaCl and 10% dextran sulfate at 55 °C with radiolabeled oligonucleotide c) of the ripening-related protein DNA.

Detection of the ribozyme-encoding gene directed against the ripening-related protein RNA. 2 small pieces of leaf from the transgenic tomato plant or from a non-transformed wild-type tomato were comminuted under liquid nitrogen with a mortar and pestle. 25 The powder was placed in Eppendorf tubes and mixed with 500 /il of 2 x CTAB buffer (2 x CTAB: 2% cetyltrimethyl-amraonium bromide, 100 mM Tris pH 8.0, 20 mM EDTA, 1.4 M NaCl, 1% polyvinylpyrrolidone MW = 40,000) which had previously been heated it 65 °C. Then 500 pi of 30 chloroform/isoamyl alcohol 24:1 (v/v) were added, and the aqueous phase was extracted. The two phases were then separated by centrifugation. The aqueous phase was pipetted into a new Eppendorf tube, and 100 p.1 of 5% CTAB which had been heated at 654C were added. Another extrac-35 tion with chloroform/isoamyl alcohol was carried out ff - '/ 235181 before 500 pi of CTAB precipitating buffer (1% CTAB, 50 mM Tris pH 8.0, 10 mM EDTA) were added to the aqueous phase. After centrifugation, the precipitate was dissolved in high-salt TE (10 mM Tris pH 8.0, 1 mM EDTA, 1 M 5 NaCl) and precipitated with 2 1/2 volumes of ethanol. After centrifugation, washing and drying, the precipitate was taken up in water and treated with 25 ng/ml RNase A (final concentration). The RNase was subsequently removed by phenol treatment. After renewed drying, the DNA was 10 taken up in 50 pi of water.

Then 4 ^g of the DNA were hydrolyzed with the restriction endonuclease Eco RI at 37°C for 1 hour. The hydrolyzate was fractionated on a 1% agarose gel. The gel was shaken with 0.4 N NaOH/O.6 M NaCl for 30 min and then with 0.5 15 M Tris CI pH 7.5/1.5 M NaCl for 30 min. The size standard used was a PstI hydrolyzate of A-phage DNA.

The DNA was then transferred to a Gene Screen Plus membrane with 10 x SSC via a capillary blot. The filter was then dried and prehybridized with 1% SDS/1 M NaCl/10% 20 dextran sulfate. For the hybridization, the prehybridiza-tion mix was mixed with a radiolabeled sample of oligonucleotide c) which had previously been boiled for 10 minutes.

The ribozyme-encoding gene was detected by the appearance 25 of blackening at about 0.8 kb on the X-ray film placed on top.

Detection of the in vitro activity of the ribozymes The oligonucleotide which encoded p. ribozyme against the ripening-related protein RNA was cloned into the Blue-30 script vector which had been opened after hydrolysis with the restriction endonucleases Xbal and PstI. In parallel with this, a DNA fragment of the ripening-related protein (nucleotide numbers 792-815 of DNA sequence published by Ray, J. et al., Nucleic Acids Res. 15, 10587 (1987)) was 29z 5 1 £■& j cloned into the Sacl/Kpnl cleavage site in the same vector. Both vectors were used in an in vitro RNA polymerase reaction as DNA templates for the RNA synthesis.

For this, in parallel, the vector carrying the ribozyme 5 gene and the vector carrying the DNA fragment of the ripening-related protein gene were cut with the restriction endonuclease Sacl. 1 samples of the opened vectors were then incubated with 10 /imol of each of the nucleotides ATP, GTP, CTP, UTP and (o-32P)UTP (5 ...) in 10 50 mM HEPES (pH 7.5) and 10 U of T7 RNA polymerase at 37°C for 30 minutes. The RNA was, after DNase treatment, precipitated in ethanol.

The synthesized RNA of the vector carrying the ribozyme gene and the RNA of the vector carrying the ripening-15 related protein gene fragment were incubated together in 50 mM Tris.CI (pH 7.8) and 10 mM MgCl2 at 25°C for 2 hours. The reaction products were then separated on a 5% denaturing polyacrylamide gel (8 M urea) and identified by autoradiography on an X-ray film. The auto-20 radiogram shows that, in the presence of ribozyme RNA, the RNA transcript of the ripening-related protein gene fragment was cleaved.

Detection of the delayed ripening of transgenic tomatoes Comparison of a non-transformed wild-type tomato plant 25 with a transgenic tomato plant which carries the ribozyme-encoding gene directed against the ripening-related protein RNA revealed that the tomatoes from the transgenic plant ripened several days later. The ribozyme activity and the delay in ripening associated therewith 30 has thus also been detected in the tomato fruit, which represents the actual site of action. ^3*181

Claims (1)

1. WHAT WE CLAIM IS: 1- a ribozyme-encoding gene or gene fragment having the dna sequence a) 5' CTAGATGATACATGCTGATGAGTCCGTGAGGACGAAACTCCATCTGCA 3' 3'TACTATGTACGACTACTCAGGCACTCCTGCTTTGAGGTAG 5' b) 5' -CTAGATTAGGTCAGCTGATGAGTCCGTGAGGACGAAACTTGCTACTGCA-3' 3'-TAATCCAGTCGACTACTCAGGCACTCCTGCTTTGAACGATG-5' C) 5 ' - CTAGACTGTATCGCTGATGAGTCCGTGAGGACGAAACAAAGCACTGCA- 3' 3 -TGACATAGCGACTACTCAGGCACTCCTGCTTTGTTTCGTG - 5' 2. RNA with ribozyme activity of the sequence 3-KKKKKKKKCA A a G in which K KKKKKKKK-5 C U CG GvA vv V V ribozyme Loop are nucleotides A, C, G or U complementary to the plant ripening enzyme RNA, are variable nucleotides A, C, G or U and are variable nucleotides A, C, G or U in the loop, where the number of VL nucleotides in the loop is a number from 0 to 550. - 16 - RNA with ribozyme activity with the sequence a) cuaccuca guacauagu c a u a g g a c gagu a u g c g c a u c g b) aucguuca a a g gacuggauu c u g a c gagu a u g c g c a u c g acgaaaca a a g gcuauguca c u g c gagu a u m 17 ? 7 c P 1 4. A process for preparing a ribozyme-encoding gene or gene fragment having the DNA sequence a) 5' CTAGATGATACATGCTGATGAGTC CGTGAGGACGAAACTCCATCTGCA 3' 3' TACTATGTACGACTACTCAGGCACTCCTGC7TTGAGGTAG 5 ' b) 5' -CTAGATTAGGTCAGCTGATGAGTCCGTGAGGACGAAACTTGCTACTGCA- 3' 3' -TAATCCAGTCGACTACTCAGGCACTCCTGCTTTGAACGATG- 5' C) 5' -CTAGACTGTATCGCTGATGAGTCCGTGAGGACGAAACAAAGCACTGCA- 3' 3' -TGACATAGCGACTACTCAGGCACTCCTGCTTTGTTTCGTG-5' by synthesis of oligonucleotides, which comprises synthesizing oligonucleotides whose initial and final sequences are each composed of 5 to 10 nucleotides which, taken together, are complementary to a DNA sequence of the ripening enzyme to be inhibited and are separated by an interpolated DNA sequence which is composed partly of specific nucleotides predetermined for the functionality of the ribozyme and partly of variable nucleotides - 5. A process as claimed in claim 4, wherein the initial and final sequences of the synthesized oligonucleotides are 7 tc 10 nucleotides. 6 . A process for preparing RNA with ribozyme activity of the sequence, 3-KKKKKKKKCA KKKKKKKK-5 A C , which comprises synthesizing an sequence S'-K'K'K'K'K'K'K'K'CTGAVGAG - 18 - 3'-KKKKKKKK GACTVCTC v' v' Vt v: vt' v; v; vJ v' vcgaaack ' k ' x' ' k ' - 3' «j L L J L v v vlvlvlvlvlvlv vgctttgk XXXXXXX-5' in which X are nucleotides A, C, G or T complementary to the plant ripening enzyme RNA, V are variable nucleotides A, C, G or T, VL are variable nucleotides A, C, G or T, where the number of VL nucleotides is a number from 0 to 550, and K't V' VL' is in each case nucleotides A, C, G or T complementary to X, V, VL, which is cloned into an intermediary vector with a plant promoter, then cloned together with the plant promoter into a binary plant vector, and a plant is transformed with the plasmid DNA obtained in this way. The process as claimed in claim 6r wherein the synthesized RNA with ribozyme activity is an RNA of the sequence a) CUACCUCA GUACAUAGU c A U A G G A C GAGU A U G C G C / * ^ A U ( G ; i41)1 ^ <i .c I ? 0 MAY 7* ii* b) aucguuca a a G - 19 - gacuggauu c U g a 9 7 q 1 o 181 c gagu A U g c g c A U C g or acgaaaca gcuauguca c c) A A g U g A c gagu A U g c g c A G U g 8. Plant cells, plants, the seeds and parts thereof, containing one or more of the DNA seqiiences as claimed in claim 1. 9- Plant cells, plants, the seeds and parts thereof, containing one or more of the RNA sequences as claimed in either claim 2 or 3. containing one or more of the DNA sequences as claimed in ,■ \ claim 1. J {20MAYI99 11. a tomato, parts thereof, plant cells or seeds thereof,- , V ^ ' r t s/ >35131 20 containing one or more of the RNA sequences as claimed in 12. The use of ribozymes for inhibiting the synthesis of ripening enzymes in plants. 13. The use as claimed in claim 12, wherein the plants are fruit-bearing plants. 14. The use as claimed in claim 13, wherein the fruit-bearing plants are tomatoes. 15. The use as claimed in one or more of claims 12 to 14, wherein the RNA sequence as claimed in either claim 2 or 3 is employed as the ribozyme 16- A gene or gene fragment according to claim 1 substantially as herein described or exemplified. 17. RNA according to claim 2 or 3 substantially as herein described or exemplified. 18. A process according to claim A substantially as herein described or exemplified. 19. A process according to claim 6 substantially as herein described or exemplified. 20. A plant cell, plant, seed or part thereof according to any one of claims 8, 9, 10 or 11 substantially as herein described or exemplified. either claim 2 or 3.