EP0683819A1 - Transgenic plants including a transgene consisting of a hybrid nucleic acid sequence, comprising at least one non-edited mitochondrial gene fragment of superior plants and process for their production - Google Patents
Transgenic plants including a transgene consisting of a hybrid nucleic acid sequence, comprising at least one non-edited mitochondrial gene fragment of superior plants and process for their productionInfo
- Publication number
- EP0683819A1 EP0683819A1 EP94906945A EP94906945A EP0683819A1 EP 0683819 A1 EP0683819 A1 EP 0683819A1 EP 94906945 A EP94906945 A EP 94906945A EP 94906945 A EP94906945 A EP 94906945A EP 0683819 A1 EP0683819 A1 EP 0683819A1
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- European Patent Office
- Prior art keywords
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- tca
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y109/00—Oxidoreductases acting on a heme group of donors (1.9)
- C12Y109/03—Oxidoreductases acting on a heme group of donors (1.9) with oxygen as acceptor (1.9.3)
- C12Y109/03001—Cytochrome-c oxidase (1.9.3.1)
Definitions
- TRANSGENIC PLANTS INCLUDING A TRANSGEN CONSISTING OF A HYBRID NUCLEIC ACID SEQUENCE, COMPRISING AT LEAST ONE FRAGMENT OF UNMITTED MITOCHONDRIAL GENE OF SUPERIOR PLANTS AND THEIR PRODUCTION METHOD.
- the present invention relates to hybrid nucleic acid sequences, comprising at least the coding region of a non-edited mitochondrial gene from higher plants and allowing the control of the male fer ⁇ tility of plants containing said sequences, to plants transgenic having such sequences, as well as a method of producing male-sterile trans ⁇ gene plants and a method of restoring male-fertile plants.
- the control of male fertility in plants is one of the key problems for obtaining hybrids, and more particularly male-sterile lines which are of agronomic interest, in particular for the control and seed improvement.
- the production of large-scale hybrid seeds, with controlled characteristics is a real challenge because many cultures have both male and female reproductive organs (stamens and pistils). This results in a significant rate of self-pollination and makes it difficult to control crosses between lines, in order to obtain the desired hybrids.
- the inventors have developed new male-sterile transgenic plants, capable of being restored and which facilitate the development of hybrid cultures. .
- SMC Male cytoplasmic sterility
- SMC is characterized by an abortion of pollen after meiosis.
- SMC is due to a nucleus-cytoplasm incompatibility, which can occur at several levels: DNA replication, transcription of genes, maturation of transcripts, translation or assembly of multiprotein complexes.
- RNAse Male-sterile plants have also been obtained by transgenosis, using a gene coding for an RNAse, under the control of an anther-specific promoter (Mariani C. et al., Nature, 1990, 347 737). This transgene, when expressed, has a toxic effect on the cell insofar as the endogenous RNAs are degraded, thus causing cell death.
- the Applicant has therefore set itself the goal of solving the problem of obtaining controlled, reliable and reproducible male-sterile transgenic plants which can be used in agronomic seed improvement programs.
- the present invention relates to transgenic plants, having in their nuclei, an expressible hybrid sequence (transgene), comprising at least one coding region of a non-edited mitochondrial gene from higher plants and a sequence capable of transferring the expressed protein. by said coding region, towards the mitochondria, which plants are characterized in that:
- the coding regions of the unedited itochondrial genes are chosen from the genes coding for a protein of the ATP synthase complex chosen from the gene fragment of 1 ⁇ TP9 from wheat, of the following formula I:
- the sequence capable of transferring said expressed protein to the mitochondria is selected from the group consisting of fragments encoding yeast tryptophanyl tRNA synthetase (SCHMITZ, UK et al., 1989, The Plant Cell, 1, 783-791), the ATPase ⁇ subunit of Nicotiana plumbaginifolia (BOUTRY et al., 1987, Nature, 328: 340-342), and the ATP / ADP translocator of corn (BATHGATE et al., 1989, Eur. J.
- said hybrid nucleic acid sequence comprises the coding region of formula I of the gene coding for the unedited form of wheat ATP9, to which is associated as a trans ⁇ sequence. fert, codons 1 to 62 of the yeast cytochrome oxidase (cox IV) subunit IV pre-sequence (SEQ. ID n ° 1).
- said acid sequence hybrid nucleic acid comprises the region fragment coding for the unedited form of 1 ⁇ TP6 of wheat, of formula II below:
- said hybrid nucleic acid sequence comprises the region fragment coding for the unedited form of cox II of formula III below:
- transgenic plants are generally selected from plants of agronomic, medical or industrial interest. More precisely, any transformable and regenerable plant can constitute the raw material for obtaining a transgenic plant in accordance with the invention.
- transformable any plant having the possibility of integrating a gene at the nuclear level, in a stable and transmissible manner, into its direct progeny.
- regenerable is understood to mean any plant having the capacity to produce neoformed plants (neoformation or micro ⁇ propagation).
- the following plants can be transformed in accordance with the invention: tobacco, rapeseed, sunflower, soy, tomato, potato, melon, carrot, chilli, endive, clover, lupine, beans, peas, corn, wheat, rye, oats, barley, rice, millet, citrus, cotton.
- the plants, from which the unedited mitochondrial genes are obtained, are selected so that the changes in nucleotides (process called editing) between the unedited sequence and the edited sequence are significant: at least 8 modified codons, and preferably at least 10 modified codons.
- the unedited mitochondrial genes are obtained, without limitation, from wheat, tobacco, petunia or potato.
- Yeast pre-sequences are, in particular, functional for the importation of proteins into the mitochondria, in plants.
- the plant from which said unedited mitochondrial gene is obtained and the plant which has incorporated the transgene may be identical or different.
- the plants transformed by such a sequence present, in at least 50% of them, a male-sterile phenotype, while not presenting any other disturbances, as regards the development of the plant. .
- transgenic plants make it possible to control, in a reliable and reproducible manner, the natural process of SMC, in particular by avoiding self-pollination, without introducing new, artificial functions into these latter and destructive, as is the case in particular in the systems described by Mariani et al. (RNAse system) or by WORRALL D. et al. (callase system).
- the present invention also relates to a hybrid nucleic acid sequence, comprising at least the coding region of a non-edited mitochondrial gene from higher plants, with which is associated a sequence capable of transferring the protein expressed by said coding region, to the mitochondrion, characterized in that:
- the coding regions of the unedited mitochondrial genes are chosen from the genes coding for a protein of the ATP synthase complex chosen from the gene fragment of 1 ⁇ TP9 from wheat, of the following formula I:
- the nucleic acid sequence capable of transferring said expressed protein to the mitochondria is selected from the group consisting of fragments coding for yeast tryptophanyl tRNA synthetase, the ⁇ subunit of ATPase from Nicotiana pl umbaginifolia, the ATP translocator / Corn ADP and an Ecorl / Kpnl fragment of 303 base pairs including codons 1 to 62 of the yeast cytochrome oxidase subunit IV, which hybrid sequence is capable of modifying the male fertility of plants having incorporated.
- said hybrid nucleic acid sequence comprises the coding region of formula I of the gene coding for the unedited form of 1 ⁇ TP9 of wheat, to which is associated as transfer sequence, codons 1 to 62 of the yeast cytochrome oxidase (cox IV) subunit IV pre-sequence (SEQ ID # 1).
- it comprises the region fragment coding for the unedited form of 1' ATP6 wheat, Formula II above, with which is associated as transfer sequence, codons 1 to 62 of the pre-sequence of the yeast cyto ⁇ chromium oxidase (cox IV) subunit IV (SEQ. ID n "3).
- hybrid nucleic acid sequence comprises the region fragment coding for the unedited form of cox II of formula III above, with which is associated as transfer sequence, the codons 1 to 62 of the pre-sequence of the yeast cyto ⁇ chromium oxidase (cox IV) subunit IV (SEQ. ID No. 5).
- a subject of the present invention is also a plasmid, characterized in that it includes a hybrid nucleic acid sequence in accordance with the invention, associated with a promoter chosen from promoters expressing constitutively and promoters s' expressing at the level of the anthers and to a suitable terminator.
- said plasmid comprises the 35S promoter and the terminator of the CaMV gene VI.
- said plasmid also comprises at least one marker gene, in particular, and this without limitation, a gene for resistance to an antibiotic and preferably, the gene for resistance to hygromycin.
- transgenic plants are likely to be obtained using a production process.
- tion of transgenic plants which comprises, for the transformation of the selected higher plant, the introduction of at least one copy of the hybrid nucleic sequence as defined above, into a receptor plant, using a plasmid containing said sequence, as defined above.
- Such a transformation can advantageously be obtained by one of the following methods: transformation of protoplasts, agrotransformation, microinjection, biolistics.
- the subject of the present invention is also a method of inhibiting the production of pollen in higher plants, characterized in that it comprises the following steps: (a) insertion of a hybrid nucleic acid sequence, such as defined above, in selected plants, by any appropriate means;
- transgenic male-sterile transgenic plants in accordance with the invention, by crossing said transgenic male-sterile plants with transgenic plants comprising in their nuclei a nucleic acid sequence.
- anti-sense hybrid that is to say including at least the same non-edited mitochondrial gene coding region of plants as that included in said male-sterile transgenic plants, in the reverse sense.
- the present invention also relates to a method of restoring male-fertile plants, from male-sterile transgenic plants, in accordance with the invention, characterized in that it comprises the following stages: (1) transformation of the higher plant selected by the introduction of at least one copy of the hybrid nucleic sequence as defined above, into a recipient plant, using a plasmid containing said sequence , for obtaining male-sterile trans ⁇ gene plants (PTMS);
- PTMS male-sterile trans ⁇ gene plants
- the present invention also relates to plasmids including a hybrid anti-sen ⁇ sequence, as defined below, associated with a promoter chosen from the constitutive promoters and the specific promoters of the anthers and also associated with a suitable terminator.
- the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention.
- sequences coding for ATP9 are obtained from a cDNA corresponding to the edited and unedited forms of wheat mitochondrial mRNA.
- L ⁇ TP9 is fused to an EcoRI / Kpnl fragment of 303 base pairs from a plasmid designated 19.4
- the resulting fragment, obtained after digestion with the enzyme HincII, is ligated at the Smal restriction site of the plasmid pDH51 (PIETRZAK et al., 1986, Nucleic Acids Re ⁇ ., 14: 5857-5868).
- the hygromycin resistance gene is inserted at the KindIII level of the plasmid pDH51, of the plasmid pEA903 (edited form of 1 ⁇ TP9, FIG. 1) and of the plasmid pEA904 (unedited form of 1 ⁇ TP9, FIG. 2) giving respective birth ⁇ ment to plasmids pH1 ( Figure 3), pH5 ( Figure 4) and pH2 ( Figure 5).
- the plasmid pH4 (FIG. 6) consists of the plasmid pEA904 in which the coding part IV / ATP9 is placed in reverse orientation by comparison with the plasmid pH2.
- sequence according to the invention can specifically be amplified using the following oligonucleotide primers:
- the unedited protein is more hydrophilic with 6 additional residues at the C-terminal level; moreover, this unedited form of ATP9 selected, consti ⁇ kills a protein model modified particularly advantageous ⁇ geous since it constitutes an element of the proton channel of ATP synthase, and accordingly, it is the function indi ⁇ pen ⁇ able of this complex; this fragment is also advantageous because of the reduced size of the coding sequence, which facilitates manipulation and the fact that 1 ⁇ TP9 can have nuclear or mitochondrial localization.
- EXAMPLE 2 Production of male-sterile transgenic plants.
- Au ⁇ i well the plasmid construction according to the invention (see example 1, pla ⁇ mide pH2) that the con ⁇ truction ⁇ controls (plasmid pHl) and the constructions ⁇ corresponding to the edited form of 1 ⁇ TP9 plasmid pH5) are used for the transformation of protoplasts of a line of Nicotiana tabacum cv. Petit Havana, called SRI.
- the protoplasts used for the transformation are isolated from the leaves of plants of Nicotiana tabacum SRI, grown in axenic condition and one month old. The young leaves are removed, removed from the central vein and cut into thin strips. The fragments are then incubated in the dark at 26 "C, overnight, in an enzy solution a- tick consisting of K3 medium (NAGY and MALIGA, 1976) supplemented with Onozuka RIO cellulase (1.2%), Onozuka RIO macerozyme (0.4%) and Fluka driselase (0.1%) (pH 5, 6). Before harvesting, the enzymatic solution is diluted with a 0.6 M sucrose solution, 0.1% MES (w / v) (pH 5.6) in the expected proportion 2v / lv.
- the protoplasts are separated from undigested tissues by filtration through a tami ⁇ of 100 ⁇ m.
- the suspension is covered with a W5 solution (MENCZEL et al., Theor. Appl. Genetics, 1981, 59: 191-195), taking care not to mix the liquid phases.
- W5 solution MENCZEL et al., Theor. Appl. Genetics, 1981, 59: 191-195
- the protoplasts are collected in the form of a band at the interface between the W5 solution and the enzymatic solution. They are collected carefully and washed twice with solution 5 to remove the traces of eny.
- These prototypes are placed in a cold room at 4-6 "C for 1-2 hours.
- 300 ⁇ l of protoplate suspension (5 ⁇ 10 6 protoplasts) are distributed in a 12 ml conical tube, then 20 ⁇ g of plasmid pH2 (or plasmid pH4), depending on the transgenic plant that one wishes to obtain, 300 ⁇ l of a solution of PEG 4000 [(PEG 4000 Merck; 40% (w / v), mannitol Merck, 0.4 M; Ca (N0 3 ) 2 4H 2 0 Merck; pH 8 (solution sterilized by filtration at 0.45 ⁇ m)] and 60 ⁇ g of DNA from calf thy ⁇ mus as carrier DNA, are added to the suspension.
- PEG 4000 (PEG 4000 Merck; 40% (w / v), mannitol Merck, 0.4 M; Ca (N0 3 ) 2 4H 2 0 Merck; pH 8 (solution sterilized by filtration at 0.45 ⁇ m)] and 60 ⁇ g of DNA from calf thy ⁇ mus as carrier DNA
- the resulting colonies are successively cultivated, in the presence of the hygro ycine selection agent at 20 mg / 1, in medium A50m (medium A containing 50 g / 1 mannitol) (CABOCHE, 1980) during the first month, then on medium A30m (medium A containing 30 g / 1 mannitol) during the second month, and finally on medium Am (medium A san ⁇ mannitol), medium containing 40 mg / ml of hyomomycin, during the third month.
- the calluses are transferred to the AR medium.
- the AR medium is medium A containing only 20 g / 1 sucrose as a carbohydrate source and 0.25 mg / 1 BAP as a growth hormone.
- the seedlings grown from cal are cultivated on medium T (NITCH and NITCH, 1969).
- the MSoo medium is used for the maintenance of plants.
- F fertile
- F / S semi-fertile
- S male-sterile average value of seed production per capsule after self-pollination.
- line H1 transgenic plants obtained with the plasmid pHl
- line H2 transgenic plants obtained with the plasmid pH2
- line H5 transgenic plants obtained with the plasmid pH5.
- SRI control line unprocessed plant.
- the size of the plants is not significantly different from that of the untransformed SRI lines.
- the average number of nodes is similar in the three different transgenic lines (19 to 24 nodes per plant).
- the transformants H1 and H5 produce fertile plants, while the transformants H2 have fertility, semi-fertility or sterility characteristics defined on the basis of pollen germination or by the reaction to fluorescein diacetate.
- the pollen viability In fertile transgenic plants, the pollen viability is between 31 and 75%, close to the value found in the SRI control line; In semi-fertile plants, the pollen viability is around 10 to 20%; in male-sterile plants, the viability is generally less than 2%.
- Plant fertility is also determined by the production of seeds after self-pollination or backcrossing. The results are also illustrated in Table I below.
- the Hl and H5 lines have an average seed production of 100 mg / capsule comparable to that of the SRI control lines (110 mg / capsule).
- the H2 lines which correspond to sterile plants produce no seeds, the semi-fertile plants produce between 12 and 50 mg / capsule, the fertile plants produce on average 100 mg / capsule. These values are well correlated with pollen viability.
- the characteristic of female fertility, for sterile and semi-fertile plants, is determined by backcrossing with the SRI lines as male parent. All male plants are fertile females and produce a normal quantity of viable seeds (63 to 92 mg / capsule), with a seed viability value greater than 77%. Thus the sterile or semi-fertile character in 50% of H2 lines is due to the absence or very low production of viable pollen.
- transgenes The transmission of transgenes is analyzed through the genetic segregation of the hygromycin phosphotransferase (hpt) gene in descendants (between 200 and 500 analyzed descendant). After self-fertilization (fertile and / or semi-fertile plants), the resistance to hygromycin is transmitted in the majority of these as a Mendelian character (mono or genetic). After retrocroi ⁇ ement with parent SRI
- the analysis of the transcription products is carried out by Southern and Northern hybridization. Total DNA was isolated from the H2 lines (sterile, semi-fertile and fertile) and H5 lines. Furthermore, the chimeric gene is analyzed by PCR amplification.
- the total DNA is isolated from 10 g of leaf tissue essentially as described in SAGHAL-MAROOF MA et al., 1984, Proc. Natl. Acad. Sci. USA, 81, 8014-8018. 1 ⁇ g of DNA is amplified in a final volume of 100 ⁇ l, using 0.5 units of polymeric Taq, 0.18 mM dNTP ⁇ and 100 pmol of each of the primers.
- the primers used are those specified in Example 1. The use of these primers excludes the amplification of endogenous TP9 (see FIG. 8C).
- RNA of the SRI, H2 and H5 lines is extracted, from the leaves, as follows: 5 g of leaves are freeze-ground; Then we proceed to a first extraction from the frozen powder, with 5 ml of a phenol: chloroform: i ⁇ oamyl alcohol mixture (25: 24: 1; v: v: v) and 5 ml of TNES + DTT (0, 1 M NaCl, 10 mM Tri ⁇ -HCl pH 7.5, 1 mM EDTA, 0.1% SDS and 2 mM dithiothreitol); we then proceed to a second extraction, starting from the phase to which, two faiths with an equal volume of chloro- form: isoamyl alcohol (24: 1; v: v) and e ⁇ t RNA precipitated with an equal volume of lithium chloride 4 M to 0 ° C overnight.
- RNAs are dissolved in water treated with DEPC. The concentration of RNA is measured by the optical density (OD) at 260 nm.
- the poly (A) + RNAs are purified by oligo (dT) -cellulose affinity chromatography. 20 ⁇ g of total RNA and 1 ⁇ g of poly (A) + RNA are subjected to electrophoresis ⁇ on 1.5% agarose gel, formaldehyde / formamide buffer, then transferred to Hybond ny ⁇ lon membranes. N + . Hybridizations with the ATP9 probe are carried out as described below.
- a 0.48 kb band was obtained with the IRS control line (FIG. 8B, part 1). This band is present in all the lines and corresponds to the endogenous mitochondrial mRNA.
- FIG. 8B shows the results obtained with the male-sterile plants H2.2 and H2.16 and with the fertile plants H5.6 and H5.15, (tracks
- the 0.98 kb transcript is absent from untransformed controls (lane 2).
- transcripts from the transgene thanks to the sequences added during the in vitro manipulation such as the regions of the pre-sequence from yeast (cox IV) and the termination region of CaMV.
- sequences added during the in vitro manipulation such as the regions of the pre-sequence from yeast (cox IV) and the termination region of CaMV.
- EXAMPLE 6 Analysis of the production of chimeric protein.
- the total RNA of the transformed plants H2 and H5 as well as the control plants were hybridized with a specific mitochondrial probe.
- transgenic protein is analyzed by immunoblotting of mitochondrial and cytosolic extracts. Antibodies directed against the fragments 21 to 54 of the pre-sequence part of cox IV of leure, forming part of the transgene, are obtained in rabbits.
- This fragment is ligated to the plasmid pGEX-A (FIG. 10) in phase with the coding sequence of the glutathione S-transferase, under the control of the promoter of ⁇ -galacto ⁇ ida ⁇ e.
- the fusion protein is induced after transformation of E. cells. coli DH5 ⁇ by IPTG. These cells produce approximately 80 mg of protein / liter of culture.
- Greenhouse plant leaves are used linked for cell fractionation. 100 ⁇ g of cyto ⁇ olic and mitochondrial proteins are fractionated by urea / SDS-PAGE.
- the immunoreaction is carried out by using an anti-cox IV anti-serum diluted to l / 500th according to the method of DARLEY-USMAR et al. [1987, Mitochondria, a practi cal approach, eds DARLEY-USMAR, (IRL Pre ⁇ Ltd.) pp. 113-152)].
- the proteins of transgenic plants carrying the male-sterile phenotype are revealed by anti-rabbit IgG antibodies conjugated to peroxidase. No signal is observed, neither with the mitochondrial fraction (FIG. 11B, lane 1), nor with the cytosolic fraction of the untransformed SRI line.
- the mitochondrial fraction of male plants - sterile H2.2 and fertile H5.15 shows a band of 12 kDa corresponding to the expected size for the protein (see Figure 11A, which specifies the structure of the 15 kDa precursor and the imported 12 kDa protein).
- the cytosolic proteins of this lineage (FIG. 11B, pi ⁇ te ⁇ 3 and 5) show 2 bands, one at
- the effect of tran ⁇ gene at the ⁇ ubcellular level must result in a dysfunction of the respiratory function of the mitochondria.
- the analysis of the breathing of the non-chlorophyllian parts of the transgenic plants was carried out.
- the respiration rates of the non-chlorophyllian organs (roots), in the presence or absence of uncouplers, are determined by analysis of the oxygen consumption using a Clark electrode. More detailed studies have been carried out on mitochondria purified by differential centrifugation and Ficoll gradient.
- the effect of decouplers on respiration and ADP / 0 ratios were determined on mitochondria from male-sterile lines and compared to transformed or wild control plants. These different measurements show that the mitochondrial function is reduced in male-sterile plants compared to the untransformed or trans ⁇ formed control with the plasmid pH5. This situation is close to that encountered in natural male-sterile plants.
- the size, the speed of growth, the number of nodes, the shape and size of the leaves and flowers are similar in transgenic plants and in control plants.
- ef ⁇ fects significatif ⁇ are observed in the male reproductive organs when the sequence of wheat ATP9 in its unedited form, is expressed in the 'tobacco plante ⁇ .
- the transformation experiments carried out with the plasmid pH2 leads to the production of tion of many plants (50%) modified in their fertility. Approximately 19% are semi-fertile and 31% are completely sterile.
- EXAMPLE 9 Construction of a chimeric gene according to the invention cox ATP6-IV (SEQ ID No. 3).
- sequences coding for ATP6 are obtained from a cDNA corresponding to the edited and unedited forms of wheat mitochondrial mRNA.
- the selected unedited ATP6 fragment has the sequence of formula II defined above and is fused to the yeast cox IV transfer sequence as defined below.
- ATP6 mRNA in wheat undergoes nucleotide changes (editing), at the level of 12 codons.
- the modifications result in the change of 11 amino acids and the loss, relative to the deduced sequence of the gene, of 7 residues, in the C-terminal region, loss caused by the creation of a stop codon.
- EXAMPLE 10 Construction of a chimeric gene according to the invention cox cox II-IV (SEQ ID No. 5).
- the coding sequences for cox II are obtained from a cDNA corresponding to the edited and unedited forms of wheat mitochondrial mRNA.
- the fragment of the unedited cox II gene has the sequence of formula III defined above and is fused to the cox IV transfer sequence of yeast as defined above.
- the resulting fragment is similar to that obtained in Example 1.
- the mRNA in wheat undergoes nucleotide changes (editing), at the level of 16 codons.
- the modifications result in the change of 16 amino acids compared to the deduced sequence of the cox gene
- CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AGA ACT TTG TGT AGC TCT 161 Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser lu 20 20
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Abstract
Hybrid nucleic acid sequences including at least the coding region of an unedited mitochondrial gene of superior plants and controlling the male fertility of plants containing said sequences, transgenic plants having such sequences and methods of production of transgenic male-sterile plants and method of restoring male-fertile plants. The nuclei of the transgenic plants contain a hybrid sequence capable of being expressed (transgene), comprising at least one coding region of an unedited mitochondrial gene of superior plants and a sequence capable of transferring the protein expressed by said coding region, to the mitochondrion, said hybrid sequence being capable of modifying the male fertility of plants having incorporated said transgene, while leaving the other phenotype characteristics of said plants unaltered.
Description
PLANTES TRANSGENIQUES INCLUANT UN TRANSGENE CONSTITUE PAR UNE SEQUENCE D'ACIDE NUCLEIQUE HYBRIDE, COMPORTANT AU MOINS UN FRAGMENT DE GENE MITOCHONDRIAL NON EDITE DE VEGETAUX SUPERIEURS ET LEUR PROCEDE DE PRODUCTION. La présente invention est relative à des sé¬ quences d'acide nucléique hybrides, comportant au moins la région codante d'un gène mitochondrial non édité de végétaux supérieurs et permettant le contrôle de la fer¬ tilité mâle des plantes contenant lesdites séquences, aux plantes transgéniques possédant de telles séquences, ainsi qu'à une méthode de production de plantes trans¬ géniques mâle-stériles et à une méthode de restauration de plantes mâle-fertiles.TRANSGENIC PLANTS INCLUDING A TRANSGEN CONSISTING OF A HYBRID NUCLEIC ACID SEQUENCE, COMPRISING AT LEAST ONE FRAGMENT OF UNMITTED MITOCHONDRIAL GENE OF SUPERIOR PLANTS AND THEIR PRODUCTION METHOD. The present invention relates to hybrid nucleic acid sequences, comprising at least the coding region of a non-edited mitochondrial gene from higher plants and allowing the control of the male fer¬ tility of plants containing said sequences, to plants transgenic having such sequences, as well as a method of producing male-sterile trans¬ gene plants and a method of restoring male-fertile plants.
Le contrôle de la fertilité mâle chez les plantes est un des problèmes clés pour l'obtention d'hy¬ brides, et plus particulièrement de lignées mâle-stériles qui présentent un intérêt sur le plan agronomique, notam¬ ment pour le contrôle et l'amélioration des semences. En effet, la production de semences hybrides à grande échelle, à caractéristiques contrôlées, est une véritable gageure parce que beaucoup de cultures présentent à la fois les organes reproducteurs mâle et femelle (étamines et pistils) . Ceci entraîne un taux important d'auto- pollinisation et rend difficile le contrôle des croise- ents entre lignées, pour l'obtention des hybrides recherchés.The control of male fertility in plants is one of the key problems for obtaining hybrids, and more particularly male-sterile lines which are of agronomic interest, in particular for the control and seed improvement. Indeed, the production of large-scale hybrid seeds, with controlled characteristics, is a real challenge because many cultures have both male and female reproductive organs (stamens and pistils). This results in a significant rate of self-pollination and makes it difficult to control crosses between lines, in order to obtain the desired hybrids.
Pour permettre l'obtention de croisements non consanguins qui permettent de produire des semences hybrides, présentant des propriétés intéressantes, les Inventeurs ont mis au point de nouvelles plantes transgé¬ niques mâle-stériles, aptes à être restaurées et qui facilitent le développement de cultures hybrides.To enable non-consanguineous crosses to be obtained which make it possible to produce hybrid seeds, having interesting properties, the inventors have developed new male-sterile transgenic plants, capable of being restored and which facilitate the development of hybrid cultures. .
La stérilité mâle cytoplasmique (SMC) se ca¬ ractérise par un avortement du pollen après la méiose. Dans les systèmes alloplasmiques, la SMC est due à une incompatibilité noyau-cytoplasme, qui peut se produire à plusieurs niveaux : réplication de l'ADN,
transcription des gènes, maturation des transcrits, tra¬ duction ou assemblage des complexes multiprotéiques.Male cytoplasmic sterility (SMC) is characterized by an abortion of pollen after meiosis. In alloplasmic systems, SMC is due to a nucleus-cytoplasm incompatibility, which can occur at several levels: DNA replication, transcription of genes, maturation of transcripts, translation or assembly of multiprotein complexes.
Des observations réalisées chez le mais et le pétunia (De ey R.E. et al., Cell, 1986, 44., 439 ; Young E.G. et al., Cell, 1987, ££, 41), émerge l'hypothèse que la SMC est due à une déficience de la machinerie bioéner¬ gétique mitochondriale. En effet, la SMC se manifeste par une réduction du taux d'ATP et de NADP. Au niveau cellu¬ laire, cette déficience est corrélée avec une dégénéres- cence des cellules du tapis de l'anthère, tout en n'ayant pas d'effet sur le développement de la plante.Observations made in corn and petunia (De ey RE et al., Cell, 1986, 44., 439; Young EG et al., Cell, 1987, ££, 41), suggests that SMC is due to a deficiency in the mitochondrial bioenergetic machinery. Indeed, SMC manifests itself by a reduction in the rate of ATP and NADP. At the cellular level, this deficiency is correlated with a degeneration of the cells of the anther carpet, while having no effect on the development of the plant.
Un certain nombre de méthodes ont été . propo¬ sées, dans l'Art antérieur, pour obtenir des plantes mâle-stériles. On peut citer notamment les rétrocroisements, qui conduisent à la substitution du génome nucléaire d'une espèce par un autre génome et ce, dans l'environne¬ ment cytoplasmique de la première espèce (alloplasmie) ; cette substitution peut également apparaître spontanément dans les cultures en champ. La SMC peut également être obtenue par fusion de protoplastes (Lonsdale D.M., Gene- tic Engineering, 1987, , 47).A number of methods have been. proposed in the prior art to obtain male-sterile plants. Mention may be made in particular of backcrosses, which lead to the substitution of the nuclear genome of one species by another genome, in the cytoplasmic environment of the first species (alloplasmia); this substitution can also occur spontaneously in field crops. SMC can also be obtained by fusion of protoplasts (Lonsdale D.M., Genetic Engineering, 1987,, 47).
Dans toutes ces situations, les résultats ne sont pas fiables, ni reproductibles ; de plus, dans tous les cas, les manipulations sont longues, fastidieuses et souvent difficiles à contrôler.In all these situations, the results are neither reliable nor reproducible; moreover, in all cases, the manipulations are long, tedious and often difficult to control.
Des plantes mâle-stériles ont également été obtenues par transgénose, à l'aide d'un gène codant pour une RNAse, sous le contrôle d'un promoteur anthère-spéci- fique (Mariani C. et al., Nature, 1990, 347. 737) . Ce transgène, lorsqu'il s'exprime, a un effet toxique sur la cellule dans la mesure où les ARNs endogènes sont dégra¬ dés, provoquant ainsi la mort cellulaire.Male-sterile plants have also been obtained by transgenosis, using a gene coding for an RNAse, under the control of an anther-specific promoter (Mariani C. et al., Nature, 1990, 347 737). This transgene, when expressed, has a toxic effect on the cell insofar as the endogenous RNAs are degraded, thus causing cell death.
Un autre système, introduisant également une nouvelle fonction artificielle et destructrice, a été dé¬ crit par orrall D. et al., (The Plant Cell, 1992, 4,
759-771) (système callase) et présente les mêmes inconvé¬ nients que le système RNAse .Another system, also introducing a new artificial and destructive function, has been described by orrall D. et al., (The Plant Cell, 1992, 4, 759-771) (callase system) and has the same disadvantages as the RNAse system.
D'autres méthodologies ont également proposées pour obtenir des plantes mâle-stérile ; on peut notamment citer les techniques qui tirent profit de la perturbation de certaines voies métaboliques (Van de Meer I.M. et al., The Plant Cell, 1992, 4, 253-262) (expression d'un gène anti-sens chalcone-synthase) ou les techniques qui font appel à l'hybridation somatique asymétrique (Melchers G. et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 6832-6836) pour mettre en présence, comme dans les lignées mâle-sté¬ riles alloplasmiques, le cytoplasme d'un individu donneur et le noyau d'un partenaire receveur. Ces deux derniers procédés présentent l'inconvénient majeur d'être très aléatoires quant à l'objectif visé, à savoir l'obtention de plantes mâle-stériles, permettant le contrôle de la reproduction de ces plantes.Other methodologies have also been proposed for obtaining male-sterile plants; include techniques which take advantage of the disruption of certain metabolic pathways (Van de Meer IM et al., The Plant Cell, 1992, 4, 253-262) (expression of an antisense gene chalcone synthase) or techniques which use asymmetric somatic hybridization (Melchers G. et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 6832-6836) to bring together, as in male-ster lines ¬ alloplasmic riles, the cytoplasm of a donor individual and the nucleus of a recipient partner. These last two methods have the major drawback of being very uncertain as to the objective sought, namely obtaining male-sterile plants, allowing the control of the reproduction of these plants.
La Demanderesse s'est, en conséquence, donné pour but de résoudre le problème de l'obtention contrô- lée, fiable et reproductible de plantes transgéniques mâle-stériles, pouvant être utilisées dans des programmes agronomiques d'amélioration de semences.The Applicant has therefore set itself the goal of solving the problem of obtaining controlled, reliable and reproducible male-sterile transgenic plants which can be used in agronomic seed improvement programs.
La présente invention a pour objet des plantes transgéniques, possédant dans leurs noyaux, une séquence hybride exprimable (transgène) , comprenant au moins une région codante d'un gène mitochondrial non édité de végé¬ taux supérieurs et une séquence susceptible de transférer la protéine exprimée par ladite région codante, vers la mitochondrie, lesquelles plantes sont caractérisées en ce que :The present invention relates to transgenic plants, having in their nuclei, an expressible hybrid sequence (transgene), comprising at least one coding region of a non-edited mitochondrial gene from higher plants and a sequence capable of transferring the expressed protein. by said coding region, towards the mitochondria, which plants are characterized in that:
- les régions codantes des gènes itochon- driaux non édités sont choisis parmi les gènes codant pour une protéine du complexe ATP synthase choisis parmi le fragment de gène de 1ΑTP9 de blé, de formule I sui- vante :the coding regions of the unedited itochondrial genes are chosen from the genes coding for a protein of the ATP synthase complex chosen from the gene fragment of 1ΑTP9 from wheat, of the following formula I:
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA
ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA ou le gène de 1ΑTP6, ou parmi les gènes codant pour une protéine de la chaîne respiratoire choisis parmi les gène des sous-unités 1 à 7 de la NAD déshydrogénase, le gène de 1 'apocytochrome b et les gènes des sous-unités I, Il ou III de la cytochrome-oxydase etATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA or the gene of 1ΑTP6, or among the genes coding for a protein of the respiratory chain chosen from the genes of subunits 1 to 7 of NAD dehydrogenase , the apocytochrome b gene and the genes of cytochrome oxidase subunits I, II or III and
- la séquence susceptible de transférer ladite protéine exprimée vers la mitochondrie est sélectionnée dans le groupe constitué par les fragments codant pour la tryptophanyl ARNt synthétase de levure (SCHMITZ, U.K. et al., 1989, The Plant Cell, 1, 783-791), la sous-unité β de l'ATPase de Nicotiana plumbaginifolia (BOUTRY et al., 1987, Nature, 328:340-342), et le translocateur ATP/ADP de mais (BATHGATE et al., 1989, Eur. J. Biochem. , 183:303-310) ou un fragment Ecorl/Kpnl de 303 paires de bases incluant les codons 1 à 62 de la sous-unité IV de la cytochrome oxydase de levure (MAARSE et al., 1984, EMBO J. , 3, 2831-2837) , laquelle séquence hybride est apte à modifier la ferti¬ lité mâle des plantes ayant incorporé ledit transgène, tout en ne modifiant pas les autres caractéristiques phé- notypiques desdites plantes.the sequence capable of transferring said expressed protein to the mitochondria is selected from the group consisting of fragments encoding yeast tryptophanyl tRNA synthetase (SCHMITZ, UK et al., 1989, The Plant Cell, 1, 783-791), the ATPase β subunit of Nicotiana plumbaginifolia (BOUTRY et al., 1987, Nature, 328: 340-342), and the ATP / ADP translocator of corn (BATHGATE et al., 1989, Eur. J. Biochem ., 183: 303-310) or an Ecorl / Kpnl fragment of 303 base pairs including codons 1 to 62 of the yeast cytochrome oxidase subunit IV (MAARSE et al., 1984, EMBO J., 3 , 2831-2837), which hybrid sequence is capable of modifying the male fertility of the plants which have incorporated said transgene, while not modifying the other phenotypic characteristics of said plants.
Selon un mode de réalisation avantageux desdites plantes transgéniques, ladite séquence d'acide nucléique hybride comprend la région codante de formule I du gène codant pour la forme non éditée de 1 'ATP9 de blé, à laquelle est associée en tant que séquence de trans¬ fert, les codons 1 à 62 de la pré-séquence de la sous- unité IV de la cytochrome-oxydase (cox IV) de levure (SEQ. ID n° 1) . Selon un autre mode de réalisation avantageux desdites plantes transgéniques, ladite séquence d'acide
nucléique hybride comprend le fragment de région codant pour la forme non éditée de 1ΑTP6 de blé, de formule II suivante :According to an advantageous embodiment of said transgenic plants, said hybrid nucleic acid sequence comprises the coding region of formula I of the gene coding for the unedited form of wheat ATP9, to which is associated as a trans¬ sequence. fert, codons 1 to 62 of the yeast cytochrome oxidase (cox IV) subunit IV pre-sequence (SEQ. ID n ° 1). According to another advantageous embodiment of said transgenic plants, said acid sequence hybrid nucleic acid comprises the region fragment coding for the unedited form of 1ΑTP6 of wheat, of formula II below:
ATG GAT AAT TTT ATC CAG AAT CTG CCT GGT GCC TAC CCG GAA ACC CCA TTG GAT CAA TTT GCC ATT ATC CCA ATA ATT GAT CTT CAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT GAA GTC TTG TAT ATG CTG CTC ACT GTC GTT TTG GTC GTT TTT CTT TTT TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCA TGG CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC CTG GTA AAC GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AAA CAA AAG TTT TTC CCT CGC ATC TCG GTC ACT TTT ACT TTT TCG TTA TTT CGT AAT CCC CAG GGT ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG GCT CTT TCA TTT TCC ATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA AGA CAT GGG CTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA CTG CCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TAT TGT TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT ATG ATG GCC GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT GCT TGG ACT ATG CTA TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GAT CTT GGT CCC TTA TTT ATA GTT CTA GCA TTA ACC GGTATG GAT AAT TTT ATC CAG AAT CTG CCT GGT GCC TAC CCG GAA ACC CCA TTG GAT CAA TTT GCC ATT ATC CCA ATA ATT GAT CTT CAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT GAA GTC TTG TAT ATG CTG CTC ACT GTC GTT TT GTC GTT TTT CTT TTT TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCA TGG CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC CTG GTA AAC GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AA TTT TTC CCT CGC ATC TCG GTC ACT TTT ACT TTT TCG TTA TTT CGT AAT CCC CAG GGT ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG GCT CTT TCA TTT TCC ATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA AGA CAT GGG CTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA CTG CCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TAT TGT TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT ATG ATG GCC GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT GCT TGG ACT ATG CTA TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GAT CTT GGT CCC TTA TTT ATA GTT CTA GCA TTA ACC GGT
CTG GAA TTA GGT GTA GCT ATA TCA CAA GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA AAT CTC CAT CAA AAT GAG TCA TTT CAT AAT TGA, à laquelle est associée en tant que séquence de trans- fert, les codons 1 à 62 de la pré-séquence de la sous- unité IV de la cytochrome-oxydase (cox IV) de levure (SEQ. ID n" 3) .CTG GAA TTA GGT GTA GCT ATA TCA CAA GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA AAT CTC CAT CAA AAT GAG TCA TTT CAT AAT TGA, to which is associated as transfer sequence, codons 1 to 62 of the yeast cytochrome oxidase (cox IV) subunit IV pre-sequence (SEQ. ID No. 3).
Selon un autre mode de réalisation avantageux deεditeε plantes transgéniques, ladite séquence d'acide nucléique hybride comprend le fragment de région codant pour la forme non éditée de cox II de formule III suivante :According to another advantageous embodiment of said transgenic plants, said hybrid nucleic acid sequence comprises the region fragment coding for the unedited form of cox II of formula III below:
ATG ATT CTT CGT TCA TTA TCA TGT CGA TTC TTC ACA ATC GCTATG ATT CTT CGT TCA TTA TCA TGT CGA TTC TTC ACA ATC GCT
CTT TGT GAT GCT GCG GAA CCA TGG CAA TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC TTA CAT CACCTT TGT GAT GCT GCG GAA CCA TGG CAA TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC TTA CAT CAC
GAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA TCA
CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT AAT CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATT CGG ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT CCA GAA GAT GAT CCA GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC AAT AGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTA ACA CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA GGT GTC AAA TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC TTC ATC TCG GTA CAA CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACT AAT CAT GCC TTT ACG CCT ATC GTC GTA GAA GCA GTG ACT TTG AAA GAT TAT GCG GAT TGG GTA TCC AAT CAA TTA ATC CTC CAA ACC AAC TAA, à laquelle est associée en tant que séquence de trans¬ fert, les codons 1 à 62 de la pré-séquence de la sous- unité IV de la cytochrome-oxydase (cox IV) de levure (SEQ. ID n° 5) . Les plantes ayant incorporé le transgène conforme à l'invention (plantes transgéniques) sont, de manière générale, sélectionnées parmi les plantes présen¬ tant un intérêt agronomique, médical ou industriel. De manière plus précise, toute plante transformable et régé- nérable peut constituer la matière première pour l'obten¬ tion d'une plante transgénique conforme à l'invention.GAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT AAT CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATT CGG ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT CCA GAA GAT CC GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC AAT AGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTA ACA CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA GGT GTC AAA TGT GAT GCT GTA CCT GGT CG TCA AAT CTT ACC TTC ATC TCG GTA CAA CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACT AAT CAT GCC TTT ACG CCT ATC GTC GTA GAA GCA GTG ACT TTG AAA GAT TAT GCG GAT TGG GTA TCC AAT CAA TTAC CTC CAA ACC AAC TAA, to which codons 1 to 62 of the yeast cytochrome oxidase (IV) subunit IV pre-sequence (SEQ. ID) are associated as a transfer sequence n ° 5). The plants which have incorporated the transgene according to the invention (transgenic plants) are generally selected from plants of agronomic, medical or industrial interest. More precisely, any transformable and regenerable plant can constitute the raw material for obtaining a transgenic plant in accordance with the invention.
Au sens de la présente invention, on entend par transformable, toute plante ayant la possibilité d'intégrer un gène au niveau nucléaire, de façon stable et transmissible, à sa descendance directe.Within the meaning of the present invention, by transformable is meant any plant having the possibility of integrating a gene at the nuclear level, in a stable and transmissible manner, into its direct progeny.
Egalement au sens de la présente invention, on entend par régénérable, toute plante ayant l'aptitude de produire des plantes néoforméeε (néoformation ou micro¬ propagation) . De manière non limitative, les plantes sui¬ vantes peuvent faire l'objet d'une transformation
conforme à 1 'invention : tabac, colza, tournesol, soja, tomate, pomme de terre, melon, carotte, piment, endive, trèfle, lupin, haricot, pois, maïs, blé, seigle, avoine, orge, riz, millet, agrumes, coton.Also within the meaning of the present invention, regenerable is understood to mean any plant having the capacity to produce neoformed plants (neoformation or micro¬ propagation). Without limitation, the following plants can be transformed in accordance with the invention: tobacco, rapeseed, sunflower, soy, tomato, potato, melon, carrot, chilli, endive, clover, lupine, beans, peas, corn, wheat, rye, oats, barley, rice, millet, citrus, cotton.
Les plantes, à partir desquelles sont obtenus les gènes mitochondriaux non édités, sont sélectionnées de manière à ce que les changements de nucléotides (processus appelé editing) entre la séquence non éditée et la séquence éditée soient importants : au moins 8 codons modifiés, et de préférence au moins 10 codons modifiés.The plants, from which the unedited mitochondrial genes are obtained, are selected so that the changes in nucleotides (process called editing) between the unedited sequence and the edited sequence are significant: at least 8 modified codons, and preferably at least 10 modified codons.
De manière préférée, les gènes mitochondriaux non édités sont obtenus, de manière non limitative, à partir du blé, du tabac, du pétunia ou de la pomme de terre.Preferably, the unedited mitochondrial genes are obtained, without limitation, from wheat, tobacco, petunia or potato.
Des pré-séquences de levures sont, en particu¬ lier, fonctionnelles pour l'importation de protéines vers la mitochondrie, chez les plantes. Conformément à l'invention, la plante à partir de laquelle est obtenu ledit gène mitochondrial non édité et la plante ayant incorporé le transgène peuvent être identiques ou différentes.Yeast pre-sequences are, in particular, functional for the importation of proteins into the mitochondria, in plants. According to the invention, the plant from which said unedited mitochondrial gene is obtained and the plant which has incorporated the transgene may be identical or different.
De manière surprenante, les plantes transfor- ées par une telle séquence présentent, chez au moins 50 % d'entre elles, un phénotype mâle-stérile, tout en ne présentant pas d'autres perturbations, en ce qui concerne le développement de la plante.Surprisingly, the plants transformed by such a sequence present, in at least 50% of them, a male-sterile phenotype, while not presenting any other disturbances, as regards the development of the plant. .
Egalement de manière surprenante, de telles plantes transgéniques permettent de contrôler, de manière fiable et reproductible, le processus naturel de la SMC, notamment en évitant l'auto-pollinisation, sans intro¬ duire dans ces dernières de fonctions nouvelles, artifi¬ cielles et destructrices, comme cela est le cas notamment dans les systèmes décrits par Mariani et al. (système RNAse) ou par WORRALL D. et al. (système callase) .
La présente invention a également pour objet une séquence d'acide nucléique hybride, comprenant au moins la région codante d'un gène mitochondrial non édité de végétaux supérieurs, à laquelle est associée une séquence susceptible de transférer la protéine exprimée par ladite région codante, vers la mitochondrie, caracté¬ risée en ce que :Also surprisingly, such transgenic plants make it possible to control, in a reliable and reproducible manner, the natural process of SMC, in particular by avoiding self-pollination, without introducing new, artificial functions into these latter and destructive, as is the case in particular in the systems described by Mariani et al. (RNAse system) or by WORRALL D. et al. (callase system). The present invention also relates to a hybrid nucleic acid sequence, comprising at least the coding region of a non-edited mitochondrial gene from higher plants, with which is associated a sequence capable of transferring the protein expressed by said coding region, to the mitochondrion, characterized in that:
- les régions codantes des gènes mitochon¬ driaux non édités sont choisis parmi les gènes codant pour une protéine du complexe ATP synthase choisis parmi le fragment de gène de 1ΑTP9 de blé, de formule I sui¬ vante :the coding regions of the unedited mitochondrial genes are chosen from the genes coding for a protein of the ATP synthase complex chosen from the gene fragment of 1ΑTP9 from wheat, of the following formula I:
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA ou le gène de 1ΑTP6, ou parmi les gènes codant pour une protéine de la chaîne respiratoire, choisis parmi les gènes des sous-unités 1 à 7 de la NAD déshydrogénase, de 1 'apocytochrome b et des sous-unités I, II ou III de la cytochrome-oxydase, etATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TG TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA or the gene of 1ΑTP6, or among the genes coding for a protein of the respiratory chain, chosen from the genes of subunits 1 to 7 of NAD dehydrogenase, of apocytochrome b and of subunits I, II or III of cytochrome oxidase, and
- la séquence nucléique susceptible de trans- férer ladite protéine exprimée vers la mitochondrie est sélectionnée dans le groupe constitué par des fragments codant pour la tryptophanyl ARNt synthétase de levure, la sous-unité β de l'ATPase de Nicotiana pl umbaginifolia , le translocateur ATP/ADP de mais et un fragment Ecorl/Kpnl de 303 paires de bases incluant les codons 1 à 62 de la sous-unité IV de la cytochrome oxydase de levure, laquelle séquence hybride est apte à modifier la ferti¬ lité mâle des plantes l'ayant incorporée.the nucleic acid sequence capable of transferring said expressed protein to the mitochondria is selected from the group consisting of fragments coding for yeast tryptophanyl tRNA synthetase, the β subunit of ATPase from Nicotiana pl umbaginifolia, the ATP translocator / Corn ADP and an Ecorl / Kpnl fragment of 303 base pairs including codons 1 to 62 of the yeast cytochrome oxidase subunit IV, which hybrid sequence is capable of modifying the male fertility of plants having incorporated.
Selon un mode de réalisation avantageux de ladite séquence d'acide nucléique hybride, elle comprend la région codante de formule I du gène codant pour la
forme non éditée de 1ΑTP9 de blé, à laquelle est asso¬ ciée en tant que séquence de transfert, les codons 1 à 62 de la pré-séquence de la sous-unité IV de la cytochrome- oxydase (cox IV) de levure (SEQ. ID n' 1) . Selon un autre mode de réalisation avantageux' de ladite séquence d'acide nucléique hybride, elle comprend le fragment de région codant pour la forme non éditée de 1 'ATP6 de blé, de formule II ci-dessus, à laquelle est associée en tant que séquence de transfert, les codons 1 à 62 de la pré-séquence de la sous-unité IV de la cyto¬ chrome-oxydase (cox IV) de levure (SEQ. ID n" 3).According to an advantageous embodiment of said hybrid nucleic acid sequence, it comprises the coding region of formula I of the gene coding for the unedited form of 1ΑTP9 of wheat, to which is associated as transfer sequence, codons 1 to 62 of the yeast cytochrome oxidase (cox IV) subunit IV pre-sequence (SEQ ID # 1). According to another advantageous embodiment 'of said hybrid nucleic acid sequence, it comprises the region fragment coding for the unedited form of 1' ATP6 wheat, Formula II above, with which is associated as transfer sequence, codons 1 to 62 of the pre-sequence of the yeast cyto¬ chromium oxidase (cox IV) subunit IV (SEQ. ID n "3).
Selon un autre mode de réalisation avantageux de ladite séquence d'acide nucléique hybride, elle comprend le fragment de région codant pour la forme non éditée de cox II de formule III ci-dessus, à laquelle est associée en tant que séquence de transfert, les codons 1 à 62 de la pré-séquence de la sous-unité IV de la cyto¬ chrome-oxydase (cox IV) de levure (SEQ. ID n' 5) .According to another advantageous embodiment of said hybrid nucleic acid sequence, it comprises the region fragment coding for the unedited form of cox II of formula III above, with which is associated as transfer sequence, the codons 1 to 62 of the pre-sequence of the yeast cyto¬ chromium oxidase (cox IV) subunit IV (SEQ. ID No. 5).
La présente invention a également pour objet, un plasmide, caractérisé en ce qu'il inclut une séquence d'acide nucléique hybride conforme à l'invention, asso¬ ciée à un promoteur choisi parmi les promoteurs s 'exprimant constitutivement et les promoteurs s'exprimant au niveau des anthères et à un terminateur convenable.A subject of the present invention is also a plasmid, characterized in that it includes a hybrid nucleic acid sequence in accordance with the invention, associated with a promoter chosen from promoters expressing constitutively and promoters s' expressing at the level of the anthers and to a suitable terminator.
Selon un mode de réalisation avantageux dudit plasmide, il comprend le promoteur 35S et le terminateur du gène VI du CaMV.According to an advantageous embodiment of said plasmid, it comprises the 35S promoter and the terminator of the CaMV gene VI.
Selon un autre mode de réalisation avantageux dudit plasmide, il comprend en outre au moins un gène marqueur, notamment, et ce de manière non limitative, un gène de résistance à un antibiotique et de préférence, le gène de résistance à 1 'hygromycine.According to another advantageous embodiment of said plasmid, it also comprises at least one marker gene, in particular, and this without limitation, a gene for resistance to an antibiotic and preferably, the gene for resistance to hygromycin.
Conformément à l'invention, les plantes trans- géniques, telles que définies ci-dessus, sont suscep¬ tibles d'être obtenues à l'aide d'un procédé de produc-
tion de plantes transgéniques qui comprend, pour la transformation du végétal supérieur sélectionné, l'intro¬ duction d'au moins une copie de la séquence nucléique hybride telle que définie ci-dessus, dans une plante réceptrice, à l'aide d'un plasmide contenant ladite séquence, tel que défini ci-dessus.In accordance with the invention, transgenic plants, as defined above, are likely to be obtained using a production process. tion of transgenic plants which comprises, for the transformation of the selected higher plant, the introduction of at least one copy of the hybrid nucleic sequence as defined above, into a receptor plant, using a plasmid containing said sequence, as defined above.
Une telle transformation peut avantageusement être obtenue par l'une des méthodes suivantes : transfor¬ mation de protoplastes, agrotransformation, microinjec- tion, biolistique.Such a transformation can advantageously be obtained by one of the following methods: transformation of protoplasts, agrotransformation, microinjection, biolistics.
La présente invention a également pour objet un procédé d'inhibition de la production de pollen, chez les végétaux supérieurs, caractérisé en ce qu'il comprend les étapes suivantes : (a) insertion d'une séquence d'acide nucléique hybride, telle que définie ci-dessus, chez les végétaux sélectionnés, par tout moyen approprié ;The subject of the present invention is also a method of inhibiting the production of pollen in higher plants, characterized in that it comprises the following steps: (a) insertion of a hybrid nucleic acid sequence, such as defined above, in selected plants, by any appropriate means;
(b) régénération et culture des végétaux transgéniques obtenus en (a) ; et (c) mesure de la production et de la viabilité du pollen (test de germination, notamment) .(b) regeneration and culture of the transgenic plants obtained in (a); and (c) measurement of pollen production and viability (germination test, in particular).
Egalement de manière surprenante, on peut res¬ taurer la fonction mâle desdites plantes transgéniques mâle-stériles, conformes à l'invention, en croisant leε- dites plantes transgéniqueε mâle-εtérileε avec deε planteε tranεgéniqueε comprenant danε leurε noyaux une séquence d'acide nucléique hybride dite anti-sens, c'est- à-dire incluant au moins la même région codante de gène mitochondrial non édité de végétaux que celle incluse dans lesditeε plantes transgéniqueε mâle-stérile, danε le εens inverse.Also surprisingly, it is possible to restore the male function of said male-sterile transgenic plants, in accordance with the invention, by crossing said transgenic male-sterile plants with transgenic plants comprising in their nuclei a nucleic acid sequence. so-called anti-sense hybrid, that is to say including at least the same non-edited mitochondrial gene coding region of plants as that included in said male-sterile transgenic plants, in the reverse sense.
La présente invention a également pour objet un procédé de restauration de plantes mâle-fertiles, à partir de plantes transgéniqueε mâle-εtérileε, conformeε à l'invention, caractérisé en ce qu'il comprend les étapes suivanteε :
(1) transformation du végétal supérieur sélec¬ tionné par l'introduction d'au moins une copie de la séquence nucléique hybride telle que définie ci-dessus, dans une plante réceptrice, à l'aide d'un plasmide conte- nant ladite séquence, pour l'obtention de plantes trans¬ géniques mâle-stérileε (PTMS) ;The present invention also relates to a method of restoring male-fertile plants, from male-sterile transgenic plants, in accordance with the invention, characterized in that it comprises the following stages: (1) transformation of the higher plant selected by the introduction of at least one copy of the hybrid nucleic sequence as defined above, into a recipient plant, using a plasmid containing said sequence , for obtaining male-sterile trans¬ gene plants (PTMS);
(2) transformation du même végétal supérieur qu'en (1), par l'introduction d'au moins une copie d'une séquence nucléique hybride anti-sens, incluant au moins la même région codante de gène mitochondrial non édité de végétaux que celle incluse dans lesdites planteε trans¬ géniques mâle-stérile obtenues en (1) , dans une plante réceptrice, à l'aide d'un plasmide contenant ladite séquence, pour l'obtention de plantes transgéniques âle- fertiles (PTMF) ;(2) transformation of the same higher plant as in (1), by the introduction of at least one copy of an antisense hybrid nucleic sequence, including at least the same coding region of mitochondrial gene not edited from plants as that included in said male-sterile trans¬ gene plants obtained in (1), in a recipient plant, using a plasmid containing said sequence, for obtaining ale-fertile transgenic plants (PTMF);
(3) croisement des planteε transgéniques mâle- stériles obtenues en (1) et des plantes mâle-fertiles ob¬ tenues en (2) , pour l'obtention d'hybrides vigoureux, restaurés danε leur fertilité mâle et présentant des caractéristiqueε présélectionnées.(3) crossing of male-sterile transgenic plants obtained in (1) and male-fertile plants obtained in (2), to obtain vigorous hybrids, restored in their male fertility and having preselected characteristics.
La présente invention a également pour objet des plasmides incluant une séquence hybride anti-senε, telle que définie ci-deεεuε, aεεociée à un promoteur choisi parmi les promoteurε constitutifs et les promo- teurs spécifiques des anthères et également associée à un terminateur convenable.The present invention also relates to plasmids including a hybrid anti-senε sequence, as defined below, associated with a promoter chosen from the constitutive promoters and the specific promoters of the anthers and also associated with a suitable terminator.
Outre les dispositions qui précèdent, l'inven¬ tion comprend encore d'autres dispositions, qui ressorti- ront de la description qui va suivre, qui se réfère à des exemples de mise en oeuvre du procédé objet de la pré¬ sente invention.In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention.
Il doit être bien entendu, toutefois, que ces exemples sont donnés uniquement à titre d'illustration de l'objet de l'invention, dont ils ne constituent en aucune manière une limitation.
EXEMPLE 1 : Construction d'un gène chimérique conforme à l'invention cox IV-ATP9 (SEQ ID n" 1) .It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation. EXAMPLE 1 Construction of a chimeric gene in accordance with the invention cox IV-ATP9 (SEQ ID No. 1).
Les séquences codant pour 1 'ATP9 sont obtenues à partir d'un ADNc correspondant aux formes éditées et non éditées d'ARNm mitochondrial de blé.The sequences coding for ATP9 are obtained from a cDNA corresponding to the edited and unedited forms of wheat mitochondrial mRNA.
LΑTP9 est fusionné à un fragment EcoRI/Kpnl de 303 paires de base à partir d'un plasmide dénommé 19.4LΑTP9 is fused to an EcoRI / Kpnl fragment of 303 base pairs from a plasmid designated 19.4
(MAARSE et al., EMBO J., 1984, 3, 2831-2837), incluant les codons 1 à 62 de la souε-unité IV ( cox IV) de la cytochrome oxydaεe de levure.(MAARSE et al., EMBO J., 1984, 3, 2831-2837), including codons 1 to 62 of the suε-unit IV (cox IV) of the yeast cytochrome oxidate.
Le fragment réεultant, obtenu aprèε digeεtion par l'enzyme HincII eεt ligaturé au niveau du εite de reεtriction Smal du plasmide pDH51 (PIETRZAK et al., 1986, Nucleic Acids Reε . , 14:5857-5868) . Le gène de réεiεtance à 1 'hygromycine eεt inεéré au niveau du εite KindIII du plasmide pDH51, du plasmide pEA903 (forme édi¬ tée de 1ΑTP9, figure 1) et du plasmide pEA904 (forme non éditée de 1ΑTP9, figure 2) donnant naissance respective¬ ment aux plasmides pHl (figure 3), pH5 (figure 4) et pH2 (figure 5) . Le plasmide pH4 (figure 6) est constitué du plasmide pEA904 danε lequel la partie codante cox IV/ATP9 eεt placée en orientation inverεe par comparaison avec le plasmide pH2.The resulting fragment, obtained after digestion with the enzyme HincII, is ligated at the Smal restriction site of the plasmid pDH51 (PIETRZAK et al., 1986, Nucleic Acids Reε., 14: 5857-5868). The hygromycin resistance gene is inserted at the KindIII level of the plasmid pDH51, of the plasmid pEA903 (edited form of 1ΑTP9, FIG. 1) and of the plasmid pEA904 (unedited form of 1ΑTP9, FIG. 2) giving respective birth ¬ ment to plasmids pH1 (Figure 3), pH5 (Figure 4) and pH2 (Figure 5). The plasmid pH4 (FIG. 6) consists of the plasmid pEA904 in which the coding part IV / ATP9 is placed in reverse orientation by comparison with the plasmid pH2.
Les séquenceε cox IV-ATP9 non-édité et cox IV- ATP9 édité sont représentées aux figures 12 et 13.The unedited cox IV-ATP9 and edited cox IV-ATP9 sequences are shown in Figures 12 and 13.
Tous ces gèneε εont εouε le contrôle du promo¬ teur CaMV 35S et du terminateur de gène VI de CaMV.All of these genes are controlled by the CaMV 35S promoter and the CaMV VI gene terminator.
Leε εéquenceε conformeε à 1 ' invention peuvent εpécifiquement être amplifiéeε à l'aide deε amorceε oli- gonucléotidiqueε suivantes :The sequence according to the invention can specifically be amplified using the following oligonucleotide primers:
(a) 5 ' -CACTACGTCAATCTATAAG-3 ' , s ' étendant du codon 3 au codon 9 de la pré-εéquence de la εous-unité IV de la cytochrome oxydase de levure et(a) 5 ′ -CACTACGTCAATCTATAAG-3 ′, extending from codon 3 to codon 9 of the pre-sequence of the yeast cytochrome oxidase IV subunit IV and
(b) 5 ' -TATGCTCAACACATGAGCG-3 ' , localisée au niveau du terminateur du gène VI du CaMV (45 paires de base en amont du signal de poiyadénylation) .
L'ARNm d'ATP9 chez le blé subit des change¬ ments de nucléotides C—U (proceεεuε appelé edi ting) , au niveau de 8 codonε. Ceε modificationε ont pour consé¬ quence le changement de 5 aminoacides dans la protéine correspondante (protéine éditée) et la perte, par rapport à la séquence déduite du gène, de 6 résidus dans la ré¬ gion C-terminale, perte provoquée par la création d'un codon stop.(b) 5 '-TATGCTCAACACATGAGCG-3', located at the terminator of the CaMV VI gene (45 base pairs upstream of the poiyadenylation signal). The ATP9 mRNA in wheat undergoes changes in nucleotides C — U (process called edi ting), at the level of 8 codons. These modifications result in the change of 5 amino acids in the corresponding protein (edited protein) and the loss, compared to the sequence deduced from the gene, of 6 residues in the C-terminal region, loss caused by the creation of a stop codon.
La protéine non éditée est plus hydrophile avec 6 résidus supplémentaires au niveau C-terminal ; de plus, cette forme non éditée d'ATP9 sélectionnée, consti¬ tue un modèle de protéine modifiée particulièrement avan- ι tageux car il constitue un élément du canal à proton de l'ATP synthase et, en conséquence, il est indiεpenεable à la fonction de ce complexe ; ce fragment eεt également avantageux en raiεon de la taille réduite de la εéquence codante, qui facilite la manipulation et le fait que 1ΑTP9 peut avoir une localiεation nucléaire ou mitochon- driale. EXEMPLE 2 : Production de plantes transgéniques mâle- stériles.The unedited protein is more hydrophilic with 6 additional residues at the C-terminal level; moreover, this unedited form of ATP9 selected, consti¬ kills a protein model modified particularly advantageous ι geous since it constitutes an element of the proton channel of ATP synthase, and accordingly, it is the function indiεpenεable of this complex; this fragment is also advantageous because of the reduced size of the coding sequence, which facilitates manipulation and the fact that 1ΑTP9 can have nuclear or mitochondrial localization. EXAMPLE 2 Production of male-sterile transgenic plants.
Auεεi bien la construction plasmidique conforme à l'invention (voir exemple 1, plaεmide pH2) que leε conεtructionε contrôles (plasmide pHl) et les constructionε correspondant à la forme éditée de 1ΑTP9 plasmide pH5) sont utiliséeε pour la transformation de protoplastes d'une lignée de Nicotiana tabacum cv. Petit Havana, dénommée SRI.Auεεi well the plasmid construction according to the invention (see example 1, plaεmide pH2) that the conεtructionε controls (plasmid pHl) and the constructionsε corresponding to the edited form of 1ΑTP9 plasmid pH5) are used for the transformation of protoplasts of a line of Nicotiana tabacum cv. Petit Havana, called SRI.
* Transformation des protoplastes : Les protoplasteε utilisés pour la transforma¬ tion sont isolés à partir des feuilles de plantes de Nicotiana tabacum SRI, cultivés en condition axénique et âgées d'un mois. Les jeunes feuilles εont prélevéeε, débarraεεéeε de la nervure centrale et coupéeε en fineε lamelleε. Leε fragmentε εont ensuite incubés à l'obscu¬ rité à 26"C, pendant une nuit, dans une solution enzy a-
tique constituée du milieu K3 (NAGY et MALIGA, 1976) additionnée de cellulase Onozuka RIO (1,2 %) , de macéro- zyme Onozuka RIO (0,4 %) et de driselase Fluka (0,1 %) (pH 5,6). Avant la récolte, la solution enzymatiσue est diluée avec une solution de saccharose 0,6 M, MES 0,1 % (p/v) (pH 5,6) dans leε proportionε reεpectiveε 2v/lv.* Transformation of the protoplasts: The protoplasts used for the transformation are isolated from the leaves of plants of Nicotiana tabacum SRI, grown in axenic condition and one month old. The young leaves are removed, removed from the central vein and cut into thin strips. The fragments are then incubated in the dark at 26 "C, overnight, in an enzy solution a- tick consisting of K3 medium (NAGY and MALIGA, 1976) supplemented with Onozuka RIO cellulase (1.2%), Onozuka RIO macerozyme (0.4%) and Fluka driselase (0.1%) (pH 5, 6). Before harvesting, the enzymatic solution is diluted with a 0.6 M sucrose solution, 0.1% MES (w / v) (pH 5.6) in the expected proportion 2v / lv.
Leε protoplastes εont εéparéε deε tissus non digérés par filtration à travers un tamiε de 100 μm. La suspension est recouverte d'une solution W5 (MENCZEL et al., Theor. Appl. Genetics, 1981, 59:191-195) en prenant soin de ne pas mélanger les phases liquides. Après cen- trifugation à 600 rpm pendant 10 min, les protoplastes sont rassemblés sous forme d'une bande à l'interface entre la solution W5 et la εolution enzymatique. Ils sont recueillis soigneuεe ent et lavéε deux fois avec la εolu¬ tion 5 pour éliminer leε traceε d'en∑y eε. Leε proto- plaεteε εont placéε danε une chambre froide à 4-6"C pen¬ dant 1-2 heures. Après une nouvelle centrifugation à 750 rpm pendant 5 min, ilε εont remiε en suspension dans une εolution mannitol/magnésium (Mannitol Merck 0,5 M ; MgCl2, 6H20 Prolabo 1,5 mM, MES Sigma 0,1 %, pH 5, 6) et leur concentration est ajustée à 1,6 x 10° proto- plaεteε/ml. Leε protoplaεtes sont soumiε à un choc ther¬ mique à 45'C pendant 5 minutes. Aprèε retour à la température ambiante, 300 μl de suspenεion de protoplaεteε (5 x 10^ protoplastes) sont répartis dans un tube conique de 12 ml. Ensuite, 20 μg de plasmide pH2 (ou de plasmide pH4) , selon la plante trans¬ génique que l'on souhaite obtenir, 300 μl d'une solution de PEG 4000 [(PEG 4000 Merck ; 40 % (p/v), mannitol Merck, 0,4 M ; Ca(N03)2 4H20 Merck ; pH 8 (εolution sté¬ rilisée par filtration à 0,45 μm) ] et 60 μg d'ADN de thy¬ mus de veau comme ADN entraîneur, sont ajoutés dans la suspenεion de protoplaεteε. Ce mélange est incubé à tem- pérature ambiante pendant 25-30 minutes, et agité douce¬ ment de temps en temps. La suεpenεion de tranεformation
est ensuite diluée progressivement en ajoutant petit à petit 10 ml de 5 sur une période de 10 minutes. Les pro- toplaεteε sont récupérés par centrifugation et repris dans 1 ml de milieu K3. * Culture des protoplastes et régénération de plantes : les protoplasteε εont cultivéε à raiεon de 5x10^ protoplastes/ml, dans 3 ml d'un mélange de milieux K3 et H (KAO et MICHAYLUK, 1975) en proportion 1:1 (v/v) , solidifié avec de l'agarose (0,8 %) . Leε colonieε résul¬ tantes sont successivement cultivées, en présence de l'agent de sélection hygro ycine à 20 mg/1, dans le milieu A50m (milieu A contenant 50 g/1 mannitol) (CABOCHE, 1980) pendant le premier mois, puis sur le milieu A30m (le milieu A contenant 30 g/1 mannitol) durant le deuxième mois, et enfin sur le milieu A-m (milieu A sanε mannitol), milieu contenant 40 mg/ml d'hy¬ gromycine, durant le troiεième moiε. Pour la régénéra¬ tion, les cals sont transféréε sur le milieu AR. Le milieu AR est le milieu A contenant seulement 20 g/1 saccharose comme source d'hydrate de carbone et 0,25 mg/1 BAP comme hormone de croiεεance. Leε plantuleε iεεueε de calε εont cultivéeε εur le milieu T (NITCH et NITCH, 1969) . Le milieu MSoo eεt utiliεé pour l'entretien deε plantes.The protoplasts are separated from undigested tissues by filtration through a tamiε of 100 μm. The suspension is covered with a W5 solution (MENCZEL et al., Theor. Appl. Genetics, 1981, 59: 191-195), taking care not to mix the liquid phases. After centrifugation at 600 rpm for 10 min, the protoplasts are collected in the form of a band at the interface between the W5 solution and the enzymatic solution. They are collected carefully and washed twice with solution 5 to remove the traces of eny. These prototypes are placed in a cold room at 4-6 "C for 1-2 hours. After a further centrifugation at 750 rpm for 5 min, they are resuspended in a mannitol / magnesium solution (Mannitol Merck 0 , 5 M; MgCl 2 , 6H 2 0 Prolabo 1.5 mM, MES Sigma 0.1%, pH 5, 6) and their concentration is adjusted to 1.6 × 10 ° protoplates / ml. The protoplates are subject to a thermal shock at 45 ° C. for 5 minutes After returning to room temperature, 300 μl of protoplate suspension (5 × 10 6 protoplasts) are distributed in a 12 ml conical tube, then 20 μg of plasmid pH2 (or plasmid pH4), depending on the transgenic plant that one wishes to obtain, 300 μl of a solution of PEG 4000 [(PEG 4000 Merck; 40% (w / v), mannitol Merck, 0.4 M; Ca (N0 3 ) 2 4H 2 0 Merck; pH 8 (solution sterilized by filtration at 0.45 μm)] and 60 μg of DNA from calf thy¬ mus as carrier DNA, are added to the suspension. This mixture is incubated at room temperature for 25-30 minutes, and stirred gently from time to time. The transformation suεpenεion is then gradually diluted by gradually adding 10 ml of 5 over a period of 10 minutes. The protoplates are recovered by centrifugation and taken up in 1 ml of K3 medium. * Culture of protoplasts and regeneration of plants: the protoplasts are cultivated at a rate of 5x10 ^ protoplasts / ml, in 3 ml of a mixture of K3 and H media (KAO and MICHAYLUK, 1975) in a 1: 1 proportion (v / v ), solidified with agarose (0.8%). The resulting colonies are successively cultivated, in the presence of the hygro ycine selection agent at 20 mg / 1, in medium A50m (medium A containing 50 g / 1 mannitol) (CABOCHE, 1980) during the first month, then on medium A30m (medium A containing 30 g / 1 mannitol) during the second month, and finally on medium Am (medium A sanε mannitol), medium containing 40 mg / ml of hyomomycin, during the third month. For regeneration, the calluses are transferred to the AR medium. The AR medium is medium A containing only 20 g / 1 sucrose as a carbohydrate source and 0.25 mg / 1 BAP as a growth hormone. The seedlings grown from cal are cultivated on medium T (NITCH and NITCH, 1969). The MSoo medium is used for the maintenance of plants.
EXEMPLE 3 : Analyse phénotypique des plantes trans¬ géniques obtenues.EXAMPLE 3 Phenotypic analysis of the transgenic plants obtained.
Les tailles des plantes âgées de 14 semaines, obtenues conformément à l'exemple 2 sont précisées dans le Tableau I ci-après :The sizes of the 14-week-old plants obtained in accordance with Example 2 are specified in Table I below:
TABLEAU I TABLE I
1 F = fertile, F/S = εemi-fertiie, S = mâle-stérile valeur moyenne de production de graine par capsule après auto-polliniεation. lignée Hl = planteε transgéniqueε obtenues avec le plasmide pHl, lignée H2 = plantes transgéniqueε obtenueε avec le plaεmide pH2, lignée H5 = plantes transgéniques obtenues avec le plasmide pH5. . lignée contrôle SRI (plante non transformée) . 1 F = fertile, F / S = semi-fertile, S = male-sterile average value of seed production per capsule after self-pollination. line H1 = transgenic plants obtained with the plasmid pHl, line H2 = transgenic plants obtained with the plasmid pH2, line H5 = transgenic plants obtained with the plasmid pH5. . SRI control line (unprocessed plant).
La taille des planteε n'est pas significative- ment différente de celle des lignéeε SRI non tranεfor- méeε. Le nombre de noeuds moyen est similaire dans les trois lignées transgéniques différentes (19 à 24 noeuds par plante) .The size of the plants is not significantly different from that of the untransformed SRI lines. The average number of nodes is similar in the three different transgenic lines (19 to 24 nodes per plant).
Apparemment, il n'y a pas de modification dans le fonctionnement des méristèmes végétatifs danε la dif¬ férenciation des noeuds et des feuilles des plantes transgéniqueε.Apparently, there is no modification in the functioning of vegetative meristems in the differentiation of nodes and leaves of transgenic plants.
La floraison dans les lignées Hl, H2 et H5 est induite 7 à 14 semaineε après la transplantation. Les fleurs des plantes tranεgéniqueε εont εimilaireε danε leurs formes et dans leurs couleurs à celles des fleurs
SRI (pétaleε roεeε-rougeε et anthèreε danε chaque fleur) . Leε planteε mâleε-εtérileε ont deε anthèrëε blancheε, contenant quelqueε grainε de pollen ou aucun (figureε 7A1 et 7B) , tandiε que leε planteε fertiles ont deε anthères blanc-jaunes avec des grains de pollen normaux (figures 7A2 et 7C) . Il n'y a aucune différence dans la forme et dans la couleur du piεtil entre les plantes mâles- εtérileε et mâleε-fertileε. EXEMPLE 4 : Analyse de la fertilité des plantes trans- géniques.Flowering in the Hl, H2 and H5 lines is induced 7 to 14 weeks after the transplant. The flowers of transgenic plants are similar in their shapes and colors to those of flowers SRI (petals of red and anther in each flower). The male-sterile plants have white anthers, containing some or no pollen grains (Figures 7A1 and 7B), while the fertile plants have white-yellow anthers with normal pollen grains (Figures 7A2 and 7C). There is no difference in the shape and color of the foot between the male-sterile and male-fertile plants. EXAMPLE 4 Analysis of the fertility of transgenic plants.
Les transformantε Hl et H5 produiεent des plantes fertiles, alors que les transformantε H2 préεen- tent deε caractéristiques de fertilité, de semi-fertilité ou de stérilité définies sur la base de la germination du pollen ou par la réaction au diacétate de fluorescéine.The transformants H1 and H5 produce fertile plants, while the transformants H2 have fertility, semi-fertility or sterility characteristics defined on the basis of pollen germination or by the reaction to fluorescein diacetate.
Dans les planteε tranεgéniqueε fertileε, la viabilité du pollen eεt compriεe entre 31 et 75 %, proche deε valeurε trouvéeε danε la lignée de contrôle SRI ; danε leε .planteε semi-fertiles, la viabilité du pollen est d'environ 10 à 20 % ; dans les plantes mâles-sté¬ riles, la viabilité est généralement inférieure à 2 %.In fertile transgenic plants, the pollen viability is between 31 and 75%, close to the value found in the SRI control line; In semi-fertile plants, the pollen viability is around 10 to 20%; in male-sterile plants, the viability is generally less than 2%.
La fertilité des plantes est également déter¬ minée par la production de graines après auto-pollinisa¬ tion ou rétrocroisement. Leε résultats sont également illustrés au Tableau I ci-desεuε.Plant fertility is also determined by the production of seeds after self-pollination or backcrossing. The results are also illustrated in Table I below.
Leε lignéeε Hl et H5 ont une production moyenne de graineε de 100 mg/capεule comparable à celle deε lignéeε contrôleε SRI (110 mg/capsule) . Les lignées H2 qui correspondent à des plantes stériles ne produisent aucune graine, les plantes semi-fertiles produisent entre 12 et 50 mg/capsule, leε planteε fertileε produiεent en moyenne 100 mg/capεule. Ces valeurs sont bien corrélées avec la viabilité pollinique.The Hl and H5 lines have an average seed production of 100 mg / capsule comparable to that of the SRI control lines (110 mg / capsule). The H2 lines which correspond to sterile plants produce no seeds, the semi-fertile plants produce between 12 and 50 mg / capsule, the fertile plants produce on average 100 mg / capsule. These values are well correlated with pollen viability.
La caractériεtique de fertilité femelle, pour les plantes εtérileε et semi-fertileε, eεt déterminée par rétrocroiεement avec leε lignéeε SRI comme parent mâle.
Toutes leε planteε mâleε-εtérileε εont fe¬ melles fertiles et produisent une quantité normale de graines viables (63 à 92 mg/capsule), avec une valeur de viabilité des graineε εupérieure à 77 %. Ainεi le carac- tère εtérile ou εemi-fertile danε 50 % deε lignéeε H2 eεt dû à l'abεence ou à la production trèε faible de pollen viable.The characteristic of female fertility, for sterile and semi-fertile plants, is determined by backcrossing with the SRI lines as male parent. All male plants are fertile females and produce a normal quantity of viable seeds (63 to 92 mg / capsule), with a seed viability value greater than 77%. Thus the sterile or semi-fertile character in 50% of H2 lines is due to the absence or very low production of viable pollen.
La transmisεion deε transgènes est analysée à travers la εégrégation génétique du gène de 1 'hygromycine phosphotransferase (hpt ) chez les descendantε (entre 200 et 500 deεcendantε analyεéε) . Aprèε auto-fécondation (planteε fertileε et/ou εemi-fertileε) , la réεiεtance à l 'hygromycine est transmiεe danε la majorité deε caε comme un caractère mendélien (mono ou di-génique) . Aprèε rétrocroiεement avec le parent SRIThe transmission of transgenes is analyzed through the genetic segregation of the hygromycin phosphotransferase (hpt) gene in descendants (between 200 and 500 analyzed descendant). After self-fertilization (fertile and / or semi-fertile plants), the resistance to hygromycin is transmitted in the majority of these as a Mendelian character (mono or genetic). After retrocroiεement with parent SRI
(plante εtérile) , 4 deε 5 planteε mâleε-εtérileε héritent du caractère de réεiεtance à l'hygromycine comme un ca¬ ractère mendélien digénique, ceci exprimant 2 loci actifε. Ceε analyεeε montrent que leε planteε stériles sont uniquement affectées dans la production de pollen, puisqu'elleε εont femelles fertiles et produiεent une quantité de graineε par fruit (100 à 150 mg) , comparable, voir supérieure à celle des témoins. EXEMPLE 5 : Analyse moléculaire des transformants.(sterile plant), 4 of 5 male sterile plants inherit the hygromycin resistance character as a digenic Mendelian character, this expressing 2 active loci. These analyzes show that the sterile plants are only affected in the production of pollen, since they are fertile females and produce a quantity of seeds per fruit (100 to 150 mg), comparable, or even greater than that of the controls. EXAMPLE 5 Molecular analysis of transformants.
Pour mettre en évidence la présence et la transcription du transgene ATP9, l'analyse des produits de transcription est effectuée par hybridation de type Southern et Northern. L'ADN total eεt isolé à partir des lignées H2 (stérileε, εemi-fertileε et fertileε) et des lignées H5. Par ailleurs, le gène chimérique eεt analyεé par amplification PCR.To demonstrate the presence and transcription of the ATP9 transgene, the analysis of the transcription products is carried out by Southern and Northern hybridization. Total DNA was isolated from the H2 lines (sterile, semi-fertile and fertile) and H5 lines. Furthermore, the chimeric gene is analyzed by PCR amplification.
* Méthodeε utiliεéeε :* Method used:
- L'ADN total eεt iεolé à partir de 10 g de tiεεuε de feuilles esεentiellement comme décrit danε SAGHAL-MAROOF M.A. et al., 1984, Proc. Natl. Acad. Sci.
USA, 81, 8014-8018. 1 μg d'ADN est amplifié dans un volume final de 100 μl, en utiliεant 0,5 unité de Taq polyméraεe, 0,18 mM dNTPε et 100 pmol de chacune deε amorceε. Leε amorceε utiliεéeε εont celleε préciεéeε à l'exemple 1. L'utiliεation de ceε amorceε exclut l'ampli¬ fication de 1ΑTP9 endogène (voir figure 8C) .- The total DNA is isolated from 10 g of leaf tissue essentially as described in SAGHAL-MAROOF MA et al., 1984, Proc. Natl. Acad. Sci. USA, 81, 8014-8018. 1 μg of DNA is amplified in a final volume of 100 μl, using 0.5 units of polymeric Taq, 0.18 mM dNTPε and 100 pmol of each of the primers. The primers used are those specified in Example 1. The use of these primers excludes the amplification of endogenous TP9 (see FIG. 8C).
L'étape de dénaturation eεt réalisée à 95"C pendant 1 min, l'étape d'hybridation est réalisée pendant 2 min à 52*C et l'étape de polymérisation est réalisée pendant 1 min à 72'C.The denaturation step eεt carried out at 95 "C for 1 min, the hybridization step is performed for 2 min at 52 ° C and the polymerization step is carried out for 1 min at 72 ° C..
25 cycles sont réalisés, les échantillons sont soumis à une électrophorèse danε un gel d'agarose à 1,5 % et transférés sur une membrane Hybond-N+ (Amersham) , comme décrit dans SAGHAL-MAROOF M.A. al. (référence citée). Les filtres sont pré-hybridés à 42'C danε de la formamide déioniεée à 50 %, 5 x SSC, 8 x Denhardt et SDS à 0,5 %. Leε filtres sont hybrides avec la séquence codante de 300 paires de base de 1ΑTP9, fragment EcoRI/HindIII marqué au 32P. Une bande (correspondant à un produit compre¬ nant 700 paires de baseε) est observée danε la plupart deε lignéeε H2 et H5 comme prévu. La figure 8A montre les résultatε obtenuε avec l'ADN H2.2 et H2.16, iεεu de planteε mâleε-εtérileε (piεteε 1 et 2) et avec deε planteε fertileε (ADN H5.6 et H5.15, piεteε 3 et 4). L'ADN issu des plantes non transformées SRI ne donne aucun signal (piste 5) .25 cycles are performed, the samples are subjected to electrophoresis in a 1.5% agarose gel and transferred to a Hybond-N + membrane (Amersham), as described in SAGHAL-MAROOF MA al. (reference cited). The filters are pre-hybridized at 42 ° C. in 50% deionized formamide, 5 × SSC, 8 × Denhardt and SDS at 0.5%. The filters are hybridized with the coding sequence of 300 base pairs of 1ΑTP9, EcoRI / HindIII fragment labeled with 32 P. A band (corresponding to a product comprising 700 base pairs) is observed in most of the lines H2 and H5 as expected. Figure 8A shows the results obtained with DNA H2.2 and H2.16, from male plant-sterile plants (parts 1 and 2) and with fertile plants (DNA H5.6 and H5.15, parts 3 and 4 ). DNA from unprocessed SRI plants gives no signal (lane 5).
- L'ARN total des lignées SRI, H2 et H5 est extrait, à partir des feuilles, comme suit : 5 g de feuilles sont cryobroyés ; puiε on procède à une première extraction à partir de la poudre congelée, avec 5 ml d'un mélange phénol:chloroforme:alcool iεoamylique (25:24:1 ; v:v:v) et 5 ml de TNES+DTT (0,1 M NaCl, 10 mM Triε-HCl pH 7,5, 1 mM EDTA, 0,1 % SDS et 2 mM dithiothréitol) ; on procède enεuite à une deuxième extraction, à partir de la phaεe àqueuεe, deux foiε avec un volume égal de chloro-
forme et d'alcool isoamylique (24:1 ; v:v) et l'ARN eεt précipité avec un volume égal de chlorure de lithium 4 M à 0*C pendant une nuit.- The total RNA of the SRI, H2 and H5 lines is extracted, from the leaves, as follows: 5 g of leaves are freeze-ground; Then we proceed to a first extraction from the frozen powder, with 5 ml of a phenol: chloroform: iεoamyl alcohol mixture (25: 24: 1; v: v: v) and 5 ml of TNES + DTT (0, 1 M NaCl, 10 mM Triε-HCl pH 7.5, 1 mM EDTA, 0.1% SDS and 2 mM dithiothreitol); we then proceed to a second extraction, starting from the phase to which, two faiths with an equal volume of chloro- form: isoamyl alcohol (24: 1; v: v) and eεt RNA precipitated with an equal volume of lithium chloride 4 M to 0 ° C overnight.
Leε ARNs sont dissous dans une eau traitée au DEPC. La concentration d'ARN est mesurée par la densité optique (DO) à 260 nm. Les ARNs poly(A)+ sont purifiés par chromatographie d'affinité oligo(dT) -cellulose. 20 μg d'ARN total et 1 μg d'ARN poly(A)+ εont soumis à une électrophorèse εur gel d'agarose 1,5 %, tampon formaldé- hyde/formamide, puis transférés sur des membranes de ny¬ lon Hybond-N+. Les hybridationε avec la sonde ATP9 sont réaliséeε comme décrit ci-deεεuε.The RNAs are dissolved in water treated with DEPC. The concentration of RNA is measured by the optical density (OD) at 260 nm. The poly (A) + RNAs are purified by oligo (dT) -cellulose affinity chromatography. 20 μg of total RNA and 1 μg of poly (A) + RNA are subjected to electrophoresis ε on 1.5% agarose gel, formaldehyde / formamide buffer, then transferred to Hybond ny¬ lon membranes. N + . Hybridizations with the ATP9 probe are carried out as described below.
Une bande de 0,48 kb eεt obtenue avec leε li¬ gnéeε contrôle SRI (figure 8B, piεte 1). Cette bande eεt présente dans toute les lignéeε et correspond à l ' ARNm endogène mitochondrial.A 0.48 kb band was obtained with the IRS control line (FIG. 8B, part 1). This band is present in all the lines and corresponds to the endogenous mitochondrial mRNA.
Un transcrit additionnel, correεpondant à une bande de 0,98 kb eεt présente seulement chez les plantes transformées. Comme illustré à la figure 8B, ces molé- cules peuvent être séparéeε de l'ARNm endogène par chro¬ matographie oligo(dT) -celluloεe, confirmant εon origine cytoplaεmique.An additional transcript, corresponding to a band of 0.98 kb, is present only in transformed plants. As illustrated in FIG. 8B, these molecules can be separated from the endogenous mRNA by oligo (dT) -cellulose chromatography, confirming its cytoplasmic origin.
La figure 8B, (piεtes 3 et 4) , montre les résultats obtenus avec les plantes mâles-εtériles H2.2 et H2.16 et avec les plantes fertileε H5.6 et H5.15, (pistesFIG. 8B, (parts 3 and 4), shows the results obtained with the male-sterile plants H2.2 and H2.16 and with the fertile plants H5.6 and H5.15, (tracks
5 et 6) . Le transcrit de 0,98 kb est absent deε contrôleε non transformés (piste 2).5 and 6). The 0.98 kb transcript is absent from untransformed controls (lane 2).
- En parallèle, par la technique de la PCR de l'ADNc, il eεt poεεible d'obtenir deε tranεcrits prove- nant du transgène grâce aux séquenceε ajoutées au cours de la manipulation in vi tro telles que les régions de la pré-séquence provenant de la levure { cox IV) et la région de terminaison du CaMV. De plus, seul le transcrit de 0,98 kb hybride avec une sonde provenant de la séquence cox IV fusionnée à 1 'ATP9.
EXEMPLE 6 : Analyse de la production de la protéine chi¬ mère.- In parallel, by the cDNA PCR technique, it is possible to obtain transcripts from the transgene thanks to the sequences added during the in vitro manipulation such as the regions of the pre-sequence from yeast (cox IV) and the termination region of CaMV. In addition, only the 0.98 kb transcript hybridized with a probe from the cox IV sequence fused to ATP9. EXAMPLE 6 Analysis of the production of chimeric protein.
Pour comprendre si leε tranεgènes affectent l'expresεion du gène mitochondrial ATP9 endogène, l'ARN total deε plantes transforméeε H2 et H5 ainεi que deε plantes contrôles ont été hybridéε avec une εonde mito- chondriale spécifique.To understand whether the transgenes affect the expression of the endogenous ATP9 mitochondrial gene, the total RNA of the transformed plants H2 and H5 as well as the control plants were hybridized with a specific mitochondrial probe.
Comme le montre la figure 9, aucune différence substantielle n'est observée lorsque le transgène est édité ou non édité et le marquage est similaire à celui du contrôle.As shown in Figure 9, no substantial difference is observed when the transgene is edited or unedited and the labeling is similar to that of the control.
La production de la protéine transgénique eεt analyεée par immunoblotting deε extraitε mitochondriaux et cytosoliques. Deε anticorpε dirigéε contre leε frag- mentε 21 à 54 de la partie pré-séquence de cox IV de le¬ vure, faisant partie du transgène, sont obtenus chez le lapin.The production of the transgenic protein is analyzed by immunoblotting of mitochondrial and cytosolic extracts. Antibodies directed against the fragments 21 to 54 of the pre-sequence part of cox IV of leure, forming part of the transgene, are obtained in rabbits.
On procède comme suit : un fragment Xbal/Kpnl contenant les codons 21 à 54 de cox IV de levure est isolé à partir du plasmide 19.4 précité.The procedure is as follows: an Xbal / KpnI fragment containing the codons 21 to 54 of yeast cox IV is isolated from the above-mentioned plasmid 19.4.
Ce fragment est ligaturé au plasmide pGEX-A (figure 10) en phase avec la séquence codante de la glu- tathion S-transférase, souε le contrôle du promoteur de la β-galactoεidaεe. La protéine de fusion est induite après transformation de cellules d'E. coli DH5α par l'IPTG. Ces cellules produisent environ 80 mg de protéines/litre de culture.This fragment is ligated to the plasmid pGEX-A (FIG. 10) in phase with the coding sequence of the glutathione S-transferase, under the control of the promoter of β-galactoεidaεe. The fusion protein is induced after transformation of E. cells. coli DH5α by IPTG. These cells produce approximately 80 mg of protein / liter of culture.
La protéine fusionnée est purifiée à partir d'un extrait d ' E. coli par chromatographie d'affinité sur une colonne de glutathion-agarose. La protéine éluée par du glutathion eεt obtenue avec un taux de pureté de l'ordre de 95 %. La protéine de fuεion eεt utilisée comme antigène pour produire des anticorps anti-cox IV chez le lapin.The fused protein is purified from an extract of E. coli by affinity chromatography on a column of glutathione-agarose. The protein eluted with glutathione is obtained with a purity of the order of 95%. The leakage protein is used as an antigen to produce anti-cox IV antibodies in rabbits.
Les feuilles de plantes de serre sont uti-
liεéeε pour le fractionnement cellulaire. 100 μg de pro- téineε cytoεoliqueε et mitochondriales sont fractionnés par urée/SDS-PAGE. L'immunoréaction est réalisée en uti¬ lisant un anti-sérum anti-cox IV dilué au l/500ème selon la méthode de DARLEY-USMAR et al. [1987, Mitochondria, a practi cal approach, eds DARLEY-USMAR, (IRL Preεε Ltd.) pp. 113-152)]. Les protéines des plantes transgéniqueε portant le phénotype mâle-stérile sont révélées par des anticorps IgG anti-lapin conjugués à de la peroxydase. Aucun signal n'est observé, ni avec la frac¬ tion mitochondriale (figure 11B, piste 1), ni avec la fraction cytosolique de la lignée SRI non tranεformée.Greenhouse plant leaves are used linked for cell fractionation. 100 μg of cytoεolic and mitochondrial proteins are fractionated by urea / SDS-PAGE. The immunoreaction is carried out by using an anti-cox IV anti-serum diluted to l / 500th according to the method of DARLEY-USMAR et al. [1987, Mitochondria, a practi cal approach, eds DARLEY-USMAR, (IRL Preεε Ltd.) pp. 113-152)]. The proteins of transgenic plants carrying the male-sterile phenotype are revealed by anti-rabbit IgG antibodies conjugated to peroxidase. No signal is observed, neither with the mitochondrial fraction (FIG. 11B, lane 1), nor with the cytosolic fraction of the untransformed SRI line.
La fraction mitochondriale deε plantes mâleε- εtérileε H2.2 et fertileε H5.15 (figure 11B, piεtes 2 et 4 respectivement) montre une bande de 12 kDa correspon¬ dant à la taille attendue pour la protéine (voir figure 11A, qui précise la εtructure du précurseur de 15 kDa et de la protéine importée de 12 kDa) .The mitochondrial fraction of male plants - sterile H2.2 and fertile H5.15 (Figure 11B, parts 2 and 4 respectively) shows a band of 12 kDa corresponding to the expected size for the protein (see Figure 11A, which specifies the structure of the 15 kDa precursor and the imported 12 kDa protein).
Les protéines cytosoliques de ceε lignéeε (figure 11B, piεteε 3 et 5) montrent 2 bandeε, l'une àThe cytosolic proteins of this lineage (FIG. 11B, piεteε 3 and 5) show 2 bands, one at
15 kDa, la taille attendue pour le polypeptide précurεeur chimérique, et l'autre à 14 kDa. La nature de ce dernier polypeptide reste à déterminer ; il s'agit probablement d'un produit de dégradation du précurseur de 15 kDa. La protéine associée avec la fraction mito¬ chondriale de la lignée H5.15 (figure 11, piste 4) migre à peu prèε au même endroit que la protéine mitochondriale H2.2, maiε légèrement en aval. Cette différence eεt due au fait que les gènes chimériques diffèrent au niveau de la position du codon stop. En effet, comme déjà préciεé ci-deεεuε, la protéine éditée présente 6 résiduε en oinε que la protéine non éditée due à la génération d'un codon εtop lorε de l'editing de l'ARN. EXEMPLE 7 : Etude de la respiration des mitochondries des plantes transgéniques.15 kDa, the expected size for the chimeric precursor polypeptide, and the other at 14 kDa. The nature of this latter polypeptide remains to be determined; it is probably a degradation product of the 15 kDa precursor. The protein associated with the mitochondrial fraction of the H5.15 line (FIG. 11, lane 4) migrates more or less to the same place as the mitochondrial protein H2.2, but may be slightly downstream. This difference is due to the fact that the chimeric genes differ at the position of the stop codon. In fact, as already mentioned below, the edited protein has 6 residues in addition to the unedited protein due to the generation of a εtop codon during editing of the RNA. EXAMPLE 7 Study of the mitochondria respiration of transgenic plants.
L'effet du tranεgène au niveau εubcellulaire
doit se traduire par un dysfonctionnement de la fonction respiratoire de la mitochondrie. L'analyse de la respira¬ tion des parties non chlorophylliennes des plantes trans¬ géniques a été réalisée. La détermination des vitesses de respiration des organes non chlorophylliens (racines) , en présence ou en absence de découplants est effectuée par analyse de la consommation d'oxygène à l'aide d'une électrode de Clark. Des études plus fines ont été conduites sur des mitochon- dries purifiées par centrifugation différentielle et en gradient de Ficoll. L'effet de découplants sur la respi¬ ration et les rapports ADP/0 ont été déterminés sur deε mitochondries issues de lignées mâles-stériles et comparé aux plantes contrôles transformées ou sauvageε. Ceε différentes mesures montrent que la fonc¬ tion mitochondriale est réduite dans les plantes mâle- stériles par rapport au témoin non transformé ou trans¬ formé avec le plasmide pH5. Cette situation est proche de celle rencontrée chez les planteε mâle-stérile natu- relies.The effect of tranεgene at the εubcellular level must result in a dysfunction of the respiratory function of the mitochondria. The analysis of the breathing of the non-chlorophyllian parts of the transgenic plants was carried out. The respiration rates of the non-chlorophyllian organs (roots), in the presence or absence of uncouplers, are determined by analysis of the oxygen consumption using a Clark electrode. More detailed studies have been carried out on mitochondria purified by differential centrifugation and Ficoll gradient. The effect of decouplers on respiration and ADP / 0 ratios were determined on mitochondria from male-sterile lines and compared to transformed or wild control plants. These different measurements show that the mitochondrial function is reduced in male-sterile plants compared to the untransformed or trans¬ formed control with the plasmid pH5. This situation is close to that encountered in natural male-sterile plants.
Il reεεort de ce qui précède que l'expreεεion dans des plantes transgéniqueε de tabac d'une séquence d'ADN codant pour de 1ΑTP9 mitochondrial non édité de blé n'a pas d'effet εur la plupart des caractères phéno- typiques des planteε tranεformées, à l'exception de l'ap¬ parition de la stérilité mâle.It follows from the above that the expression in transgenic tobacco plants of a DNA sequence coding for unpublished mitochondrial 1ΑTP9 from wheat has no effect on most of the phenotypic characters of the transformed plants. , with the exception of the appearance of male sterility.
En effet, la taille, la viteεεe de croiεεance, le nombre de noeuds, la forme et la taille des feuilles et des fleurs εont εimilaireε chez les planteε transgé- niques et chez les plantes contrôles. Toutefois, des ef¬ fets significatifε sont observés au niveau des organes de la reproduction mâle lorsque la séquence d'ATP9 de blé, sous sa forme non éditée, est exprimée dans les ' planteε de tabac. En fait, leε expérimentationε de tranεforma- tionε réalisées avec le plasmide pH2 conduit à la produc-
tion de beaucoup de plantes (50 %) modifiées danε leur fertilité. Approximativement 19 % εont εemi-fertileε et 31 % sont entièrement stériles.Indeed, the size, the speed of growth, the number of nodes, the shape and size of the leaves and flowers are similar in transgenic plants and in control plants. However, ef¬ fects significatifε are observed in the male reproductive organs when the sequence of wheat ATP9 in its unedited form, is expressed in the 'tobacco planteε. In fact, the transformation experiments carried out with the plasmid pH2 leads to the production of tion of many plants (50%) modified in their fertility. Approximately 19% are semi-fertile and 31% are completely sterile.
Toutes les lignées semi-fertileε et stériles H2 expriment le transgène sous la forme d'ARNm polyadé- nylé. Les lignées. H2 fertiles ne présentent pas le trans¬ crit de 0,98 kb, même lorsque le transgène est détecté après amplification par PCR, ceci indiquant que dans ce dernier cas le transgène est inactif. Deε réεultats montrent également, de manière inattendue, que le phénotype mâle-stérile est corrélé uniquement avec la présence de séquence non éditée d'ATP9 tandis que les transformants obtenus avec la forme éditée d'ATP9 sont tous fertiles. Dans tous les cas, les plantes stériles sont seulement mâles-stériles et peuvent être pollinisées avec un pollen étranger, ceci reflétant une fertilité femelle normale. EXEMPLE 8 : Production des planteε transgéniques présen- tant une séquence hybride conforme à l'invention anti¬ sens.All semi-fertile and sterile H2 lines express the transgene in the form of polyadenylated mRNA. The lines. Fertile H2 do not show the 0.98 kb trans¬ crit, even when the transgene is detected after amplification by PCR, this indicating that in the latter case the transgene is inactive. These results also show, unexpectedly, that the male-sterile phenotype is correlated only with the presence of unedited ATP9 sequence while the transformants obtained with the edited form of ATP9 are all fertile. In all cases, sterile plants are only male-sterile and can be pollinated with foreign pollen, this reflecting normal female fertility. EXAMPLE 8 Production of transgenic plants having a hybrid sequence in accordance with the anti-sense invention.
On procède comme danε l'exemple 2, la trans¬ formation de protoplaεtes étant toutefois réalisée à 1 'aide des plasmideε pH4. En croiεant ceε planteε mâle-fertileε avec leε planteε transgéniques mâle-stériles, conformes à l'inven¬ tion, on obtient des hybrides mâle-fertiles non consan¬ guins. EXEMPLE 9 : Construction d'un gène chimérique conforme à l'invention cox IV-ATP6 (SEQ ID n* 3).The procedure is as in Example 2, the transformation of protoplates being however carried out using the plasmids pH4. By crossing this male-fertile plant with male-sterile transgenic plants, in accordance with the invention, male-fertile hybrids are not obtained. EXAMPLE 9: Construction of a chimeric gene according to the invention cox ATP6-IV (SEQ ID No. 3).
Les séquence codant pour 1 'ATP6 sont obtenueε à partir d'un ADNc correεpondant aux formeε éditéeε et non éditéeε d'ARNm mitochondrial de blé.The sequences coding for ATP6 are obtained from a cDNA corresponding to the edited and unedited forms of wheat mitochondrial mRNA.
Le fragment d'ATP6 non édité sélectionné pré- sente la séquence de formule II définie ci-desεuε et est fusionné à la séquence de transfert cox IV de levure
telle que définie ci-deεεuε.The selected unedited ATP6 fragment has the sequence of formula II defined above and is fused to the yeast cox IV transfer sequence as defined below.
Le fragment résultant est similaire à celui obtenu à l'exemple 1.The resulting fragment is similar to that obtained in Example 1.
L'ARNm d'ATP6 chez le blé subit des change- ents de nucléotideε { edi ting) , au niveau de 12 codons. Les modifications ont pour conséquence le changement de 11 aminoacides et la perte, par rapport à la séquence déduite du gène, de 7 résiduε, danε la région C- terminale, perte provoquée par la création d'un codon stop.ATP6 mRNA in wheat undergoes nucleotide changes (editing), at the level of 12 codons. The modifications result in the change of 11 amino acids and the loss, relative to the deduced sequence of the gene, of 7 residues, in the C-terminal region, loss caused by the creation of a stop codon.
EXEMPLE 10 : Construction d'un gène chimérique conforme à l'invention cox IV-cox II (SEQ ID n* 5).EXAMPLE 10: Construction of a chimeric gene according to the invention cox cox II-IV (SEQ ID No. 5).
Les séquence codant pour cox II sont obtenues à partir d'un ADNc correspondant aux formes éditées et non éditées d'ARNm mitochondrial de blé.The coding sequences for cox II are obtained from a cDNA corresponding to the edited and unedited forms of wheat mitochondrial mRNA.
Le fragment du gène cox II non édité présente la séquence de formule III définie ci-dessuε et est fusionné à la séquence de transfert cox IV de levure telle que définie ci-dessuε. Le fragment résultant est similaire à celui obtenu à 1 ' exemple 1.The fragment of the unedited cox II gene has the sequence of formula III defined above and is fused to the cox IV transfer sequence of yeast as defined above. The resulting fragment is similar to that obtained in Example 1.
L'ARNm chez le blé subit des changements de nucléotides (editing) , au niveau de 16 codonε . Les modifications ont pour conséquence le changement de 16 aminoacides par rapport à la séquence déduite du gène coxThe mRNA in wheat undergoes nucleotide changes (editing), at the level of 16 codons. The modifications result in the change of 16 amino acids compared to the deduced sequence of the cox gene
II.II.
Ainsi que cela ressort de ce qui précède, l'invention ne se limite nullement à ceux de seε modeε de mise en oeuvre, de réalisation et d'application qui vien- nent d'être décrits de façon plus explicite ; elle en em¬ brasse au contraire toutes les varianteε qui peuvent ve¬ nir à l'eεprit du technicien en la matière, εanε ε' écar¬ ter du cadre, ni de la portée de la préεente invention.
LISTE DE SEQUENCESAs is apparent from the above, the invention is in no way limited to those of its modes of implementation, production and application which have just been described more explicitly; on the contrary, it embraces all the variants which can come to the mind of the technician in the matter, without going beyond the framework or the scope of the present invention. LIST OF SEQUENCES
( 1 ) INFORMATION GENERALE :(1) GENERAL INFORMATION:
( i ) DEPOSANT :(i) DEPOSITOR:
(A) NOM: Centre National de la Recherche Scientifique(A) NAME: National Center for Scientific Research
C.N.R.S.C.N.R.S.
(B) RUE: 3 rue Michel-Ange(B) STREET: 3 rue Michel-Ange
(C) VILLE: Paris(C) CITY: Paris
(E) PAYS: FRANCE(E) COUNTRY: FRANCE
(F) CODE POSTAL: 75016(F) POSTAL CODE: 75016
(ii) TITRE DE L' INVENTION: PLANTES TRANSGENIQUES INCLUANT UNE SEQUENCE D'ACIDE NUCLEIQUE HYBRIDE, COMPORTANT UN FRAGMENT DE GENE MITOCHONDRIAL NON EDITE DE VEGETAUX SUPERIEURS ET LEUR PROCEDE D'OBTENTION.(ii) TITLE OF THE INVENTION: TRANSGENIC PLANTS INCLUDING A SEQUENCE OF HYBRID NUCLEIC ACID, COMPRISING A FRAGMENT OF NON-EDITED MITOCHONDRIAL GENE OF SUPERIOR PLANTS AND THEIR PROCESS FOR OBTAINING SAME.
(iii) NOMBRE DE SEQUENCES: 6(iii) NUMBER OF SEQUENCES: 6
(iv) FORME LISIBLE PAR ORDINATEUR:(iv) COMPUTER-READABLE FORM:
(A) TYPE DE SUPPORT: Floppy disk(A) TYPE OF SUPPORT: Floppy disk
(B) ORDINATEUR: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) SYSTEME D' EXPLOITATION: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) LOGICIEL: Patentln Release #1.0, Version #1.25 (OEB)(D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (EPO)
(2) INFORMATION POUR LA SEQ ID NO: 1:(2) INFORMATION FOR SEQ ID NO: 1:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 568 paires de bases(A) LENGTH: 568 base pairs
(B) TYPE: acide nucléique(B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
( i i ) TYPE DE MOLECULE : ADNc pour ARNm(i i) TYPE OF MOLECULE: cDNA for mRNA
( ix ) CARACTERISTIQUE ADDITIONELLE :(ix) ADDITIONAL FEATURE:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT: 99..527(B) LOCATION: 99..527
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 1:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC ATTTCGATTT 60GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC ATTTCGATTT 60
TGATGTTGCC ATACAAATAG ATAACAAGCA CAAGCACA ATG CTT TCA CTA CGT 113TGATGTTGCC ATACAAATAG ATAACAAGCA CAAGCACA ATG CTT TCA CTA CGT 113
Met Leu Ser Leu Arg 1 5Met Leu Ser Leu Arg 1 5
CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AGA ACT TTG TGT AGC TCT 161 Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser lu 15 20
AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AAA ACT GCC CAA AAC TTA 209CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AGA ACT TTG TGT AGC TCT 161 Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser lu 20 20 AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AAA ACT GCC CAA AAC TTA 209
Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys Thr Ala Gin Asn LeuArg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys Thr Ala Gin Asn Leu
25 30 3525 30 35
GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GGT CCT GGT GCT AAA GAG 257GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GGT CCT GGT GCT AAA GAG 257
Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys Glu 40 45 50Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys Glu 40 45 50
GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG TTA GAA GGT GCT AAA TCA 305GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG TTA GAA GGT GCT AAA TCA 305
Gly Thr Arg Gly Ser Ser Arg Val Glu Met Leu Glu Gly Ala Lys Ser 55 60 65Gly Thr Arg Gly Ser Ser Arg Val Glu Met Leu Glu Gly Ala Lys Ser 55 60 65
ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT 353ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT 353
Ile Gly Ala Gly Ala Ala Thr Ile Ala Leu Ala Gly Ala Ala Val Gly 70 75 80 85Ile Gly Ala Gly Ala Ala Thr Ile Ala Leu Ala Gly Ala Ala Val Gly 70 75 80 85
ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA 401ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA 401
Ile Gly Asn Val Leu Ser Ser Leu Ile His Ser Val Ala Arg Asn Pro 90 95 100Ile Gly Asn Val Leu Ser Ser Leu Ile His Ser Val Ala Arg Asn Pro 90 95 100
TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC 449TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC 449
Ser Leu Ala Lys Gin Ser Phe Gly Tyr Ala Ile Leu Gly Phe Ala LeuSer Leu Ala Lys Gin Ser Phe Gly Tyr Ala Ile Leu Gly Phe Ala Leu
105 110 115105 110 115
ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA 497ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA 497
Thr. Glu Ala Ile Ala Leu Phe Ala Pro Met Met Ala Phe Leu Ile Ser 120 125 130Thr. Glu Ala Ile Ala Leu Phe Ala Pro Met Met Ala Phe Leu Ile Ser 120 125 130
TTC GTT TTC CGA TCG CAT AAA AAG TCA TGAGATCAAA AAAGAAATGT 544TTC GTT TTC CGA TCG CAT AAA AAG TCA TGAGATCAAA AAAGAAATGT 544
Phe Val Phe Arg Ser His Lys Lys Ser 135 ' 140Phe Val Phe Arg Ser His Lys Lys Ser 135 '140
GTGAATGTAG TTACAGATGT CGAC 568GTGAATGTAG TTACAGATGT CGAC 568
(2) INFORMATION POUR LA SEQ ID NO: 2:(2) INFORMATION FOR SEQ ID NO: 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 142 acides aminés(A) LENGTH: 142 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: protéine(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Met Leu Ser Leu Arg Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15Met Leu Ser Leu Arg Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15
Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys 20 25 30
Thr Ala Gin Asn Leu Ala Glu Val Asn Gly Pro Glu Thr Leu Ile GlyThr Leu Cys Ser Ser Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys 20 25 30 Thr Ala Gin Asn Leu Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly
35 40 4535 40 45
Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Ser Arg Val Glu Met LeuPro Gly Ala Lys Glu Gly Thr Arg Gly Ser Ser Arg Val Glu Met Leu
50 55 6050 55 60
Glu Gly Ala Lys Ser Ile Gly Ala Gly Ala Ala Thr Ile Ala Leu AlaGlu Gly Ala Lys Ser Ile Gly Ala Gly Ala Ala Thr Ile Ala Leu Ala
65 70 75 8065 70 75 80
Gly Ala Ala Val Gly Ile Gly Asn Val Leu Ser Ser Leu Ile His Ser 85 90 95Gly Ala Ala Val Gly Ile Gly Asn Val Leu Ser Ser Leu Ile His Ser 85 90 95
Val Ala Arg Asn Pro Ser Leu Ala Lys Gin Ser Phe Gly Tyr Ala IleVal Ala Arg Asn Pro Ser Leu Ala Lys Gin Ser Phe Gly Tyr Ala Ile
100 105 110100 105 110
Leu Gly Phe Ala Leu Thr Glu Ala Ile Ala Leu Phe Ala Pro Met MetLeu Gly Phe Ala Leu Thr Glu Ala Ile Ala Leu Phe Ala Pro Met Met
115 120 125115 120 125
Ala Phe Leu Ile Ser Phe Val Phe Arg Ser His Lys Lys Ser 130 135 140Ala Phe Leu Ile Ser Phe Val Phe Arg Ser His Lys Lys Ser 130 135 140
(2) INFORMATION POUR LA SEQ ID NO: 3:(2) INFORMATION FOR SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 1106 paires de bases(A) LENGTH: 1106 base pairs
(B) TYPE: acide nucléique(B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc oour ARNm(ii) TYPE OF MOLECULE: cDNA or mRNA
!ix) CARACTERISTIQUE ADDITIONELLE:! ix) ADDITIONAL FEATURE:
(A) NOM/CLE: CDΞ(A) NAME / KEY: CDΞ
(B) EMPLACEMENT: 99..1106(B) LOCATION: 99..1106
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC ATTTCGATTT 60GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC ATTTCGATTT 60
TGATGTTGCC ATACAAATAG ATAACAAGCA CAAGCACA ATG CTT TCA CTA CGT 113TGATGTTGCC ATACAAATAG ATAACAAGCA CAAGCACA ATG CTT TCA CTA CGT 113
Met Leu Ser Leu Arg 1 5Met Leu Ser Leu Arg 1 5
CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AGA ACT TTG TGT AGC TCT 161 Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser 10 15 20CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AGA ACT TTG TGT AGC TCT 161 Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser 10 15 20
AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AAA ACT GCC CAA AAC TTA 209 Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys Thr Ala Gin Asn Leu 25 30 35
GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GGT CCT GGT GCT AAA GAG 257AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AAA ACT GCC CAA AAC TTA 209 Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys Thr Ala Gin Asn Leu 25 30 35 GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GGT CCT GGT GCT AAA GAG 257
Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys GluAla Glu Val Asn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys Glu
40 45 5040 45 50
GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG GAT AAT TTT ATC CAG AAT 305GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG GAT AAT TTT ATC CAG AAT 305
Gly Thr Arg Gly Ser Ser Arg Val Glu Met Asp Asn Phe Ile Gin AsnGly Thr Arg Gly Ser Ser Arg Val Glu Met Asp Asn Phe Ile Gin Asn
55 60 6555 60 65
CTG CCT GGT GCC TAC CCG GAA ACC CCA TTG GAT CAA TTT GCC ATT ATC 353CTG CCT GGT GCC TAC CCG GAA ACC CCA TTG GAT CAA TTT GCC ATT ATC 353
Leu Pro Gly Ala Tyr Pro Glu Thr Pro Leu Asp Gin Phe Ala Ile IleLeu Pro Gly Ala Tyr Pro Glu Thr Pro Leu Asp Gin Phe Ala Ile Ile
70 75 80 8570 75 80 85
CCA ATA ATT GAT CTT CAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT 401CCA ATA ATT GAT CTT CAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT 401
Pro Ile Ile Asp Leu His Val Gly Asn Phe Tyr Leu Ser Phe Thr Asn 90 95 100Pro Ile Ile Asp Leu His Val Gly Asn Phe Tyr Leu Ser Phe Thr Asn 90 95 100
GAA GTC TTG TAT ATG CTG CTC ACT GTC GTT TTG GTC GTT TTT CTT TTT 449GAA GTC TTG TAT ATG CTG CTC ACT GTC GTT TTG GTC GTT TTT CTT TTT 449
Glu Val Leu Tyr Met Leu Leu Thr Val Val Leu Val Val Phe Leu PheGlu Val Leu Tyr Met Leu Leu Thr Val Val Leu Val Val Phe Leu Phe
105 110 115105 110 115
TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCA TGG 497TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCA TGG 497
Phe Val Val Thr Lys Lys Gly Gly Gly Lys Ser Val Pro Asn Ala TrpPhe Val Val Thr Lys Lys Gly Gly Gly Lys Ser Val Pro Asn Ala Trp
120 125 130120 125 130
CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC CTG GTA AAC 545CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC CTG GTA AAC 545
Gin Ser Leu Val Glu Leu Ile Tyr Asp Phe Val Leu Asn Leu Val AsnGin Ser Leu Val Glu Leu Ile Tyr Asp Phe Val Leu Asn Leu Val Asn
135 140 145135 140 145
GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AAA CAA AAG TTT TTC CCT 593GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AAA CAA AAG TTT TTC CCT 593
Glu Gin Ile Gly Gly Leu Ser Gly Asn Val Lys Gin Lys Phe Phe ProGlu Gin Ile Gly Gly Leu Ser Gly Asn Val Lys Gin Lys Phe Phe Pro
150 155 160 165150 155 160 165
CGC ATC TCG GTC ACT TTT ACT TTT TCG TTA TTT CGT AAT CCC CAG GGT 641CGC ATC TCG GTC ACT TTT ACT TTT TCG TTA TTT CGT AAT CCC CAG GGT 641
Arg Ile Ser Val Thr Phe Thr Phe Ser Leu Phe Arg Asn Pro Gin Gly 170 175 180Arg Ile Ser Val Thr Phe Thr Phe Ser Leu Phe Arg Asn Pro Gin Gly 170 175 180
ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG 689ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG 689
Met Ile Pro Phe Ser Phe Thr Val Thr Ser His Phe Leu Ile Thr LeuMet Ile Pro Phe Ser Phe Thr Val Thr Ser His Phe Leu Ile Thr Leu
185 190 195185 190 195
GCT CTT TCA TTT TCC ATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA 737GCT CTT TCA TTT TCC ATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA 737
Ala Leu Ser Phe Ser Ile Phe Ile Gly Ile Thr Ile Val Gly Phe GinAla Leu Ser Phe Ser Ile Phe Ile Gly Ile Thr Ile Val Gly Phe Gin
200 205 210200 205 210
AGA CAT GGG CTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA 785AGA CAT GGG CTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA 785
Arg His Gly Leu His Phe Phe Ser Phe Leu Leu Pro Ala Gly Val ProArg His Gly Leu His Phe Phe Ser Phe Leu Leu Pro Ala Gly Val Pro
215 220 225215 220 225
CTG CCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TAT TGT 833CTG CCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TAT TGT 833
Leu Pro Leu Ala Pro Phe Leu Val Leu Leu Glu Leu Ile Ser Tyr CysLeu Pro Leu Ala Pro Phe Leu Val Leu Leu Glu Leu Ile Ser Tyr Cys
230 235 240 245
TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT ATG ATG GCC 881230 235 240 245 TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT ATG ATG GCC 881
Phe Arg Ala Leu Ser Leu Gly Ile Arg Leu Phe Ala Asn Met Met Ala 250 255 260Phe Arg Ala Leu Ser Leu Gly Ile Arg Leu Phe Ala Asn Met Met Ala 250 255 260
GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT GCT TGG ACT ATG CTA 929 Gly His Ser Leu Val Lys Ile Leu Ser Gly Phe Ala Trp Thr Met Leu 265 270 275GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT GCT TGG ACT ATG CTA 929 Gly His Ser Leu Val Lys Ile Leu Ser Gly Phe Ala Trp Thr Met Leu 265 270 275
TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GAT CTT GGT CCC TTA TTT 977 Phe Leu Asn Asn Ile Phe Tyr Phe Ile Gly Asp Leu Gly Pro Leu Phe 280 285 290TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GAT CTT GGT CCC TTA TTT 977 Phe Leu Asn Asn Ile Phe Tyr Phe Ile Gly Asp Leu Gly Pro Leu Phe 280 285 290
ATA GTT CTA GCA TTA ACC GGT CTG GAA TTA GGT GTA GCT ATA TCA CAA 1025 Ile Val Leu Ala Leu Thr Gly Leu Glu Leu Gly Val Ala Ile Ser Gin 295 300 305ATA GTT CTA GCA TTA ACC GGT CTG GAA TTA GGT GTA GCT ATA TCA CAA 1025 Ile Val Leu Ala Leu Thr Gly Leu Glu Leu Gly Val Ala Ile Ser Gin 295 300 305
GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA 1073 Ala His Val Ser Thr Ile Ser Ile Cys Ile Tyr Leu Asn Asp Ala Thr 310 315 320 325GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA 1073 Ala His Val Ser Thr Ile Ser Ile Cys Ile Tyr Leu Asn Asp Ala Thr 310 315 320 325
AAT CTC CAT CAA AAT GAG TCA TTT CAT AAT TGA 1106AAT CTC CAT CAA AAT GAG TCA TTT CAT AAT TGA 1106
A.sn Leu His Gin Asn Glu Ser Phe His Asn 330 335A.sn Leu His Gin Asn Glu Ser Phe His Asn 330 335
(2) INFORMATION POUR LA SEQ ID NO: 4:(2) INFORMATION FOR SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 335 acides aminés(A) LENGTH: 335 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: protéine(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Met Leu Ser Leu Arg Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15Met Leu Ser Leu Arg Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15
Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys 20 25 30Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys 20 25 30
Thr Ala Gin Asn Leu Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly 35 40 45Thr Ala Gin Asn Leu Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly 35 40 45
Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Ser Arg Val Glu Met Asp 50 Ξ5 60Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Ser Arg Val Glu Met Asp 50 Ξ5 60
Asn Phe Ile Gin Asn Leu Pro Gly Ala Tyr Pro Glu Thr Pro Leu Asp 65 70 75 80Asn Phe Ile Gin Asn Leu Pro Gly Ala Tyr Pro Glu Thr Pro Leu Asp 65 70 75 80
Gin Phe Ala Ile Ile Pro Ile Ile Asp Leu His Val Gly Asn Phe Tyr 85 90 95
Leu Ser Phe Thr Asn Glu Val Leu Tyr Met Leu Leu Thr Val Val Leu 100 105 110Gin Phe Ala Ile Pro Ile Ile Asp Leu His Val Gly Asn Phe Tyr 85 90 95 Leu Ser Phe Thr Asn Glu Val Leu Tyr Met Leu Leu Thr Val Val Leu 100 105 110
Val Val Phe Leu Phe Phe Val Val Thr Lys Lys Gly Gly Gly Lys Ser 115 120 125Val Val Phe Leu Phe Phe Val Val Thr Lys Lys Gly Gly Gly Gly Lys Ser 115 120 125
Val Pro Asn Ala Trp Gin Ser Leu Val Glu Leu Ile Tyr Asp Phe Val 130 135 140Val Pro Asn Ala Trp Gin Ser Leu Val Glu Leu Ile Tyr Asp Phe Val 130 135 140
Leu Asn Leu Val Asn Glu Gin Ile Gly Gly Leu Ser Gly Asn Val Lys 145 150 155 160Leu Asn Leu Val Asn Glu Gin Ile Gly Gly Leu Ser Gly Asn Val Lys 145 150 155 160
Gin Lys Phe Phe Pro Arg Ile Ser Val Thr Phe Thr Phe Ser Leu Phe 165 170 175Gin Lys Phe Phe Pro Arg Ile Ser Val Thr Phe Thr Phe Ser Leu Phe 165 170 175
Arg Asn Pro Gin Gly Met Ile Pro Phe Ser Phe Thr Val Thr Ser His 180 185 190Arg Asn Pro Gin Gly Met Ile Pro Phe Ser Phe Thr Val Thr Ser His 180 185 190
Phe Leu Ile Thr Leu Ala Leu Ser Phe Ser Ile Phe Ile Gly Ile Thr 195 200 205Phe Leu Ile Thr Leu Ala Leu Ser Phe Ser Ile Phe Ile Gly Ile Thr 195 200 205
Ile Val Gly Phe Gin Arg His Gly Leu His Phe Phe Ser Phe Leu Leu 210 215 220Ile Val Gly Phe Gin Arg His Gly Leu His Phe Phe Ser Phe Leu Leu 210 215 220
Pro Ala Gly Val Pro Leu Pro Leu Ala Pro Phe Leu Val Leu Leu Glu 225 230 235 240Pro Ala Gly Val Pro Leu Pro Leu Ala Pro Phe Leu Val Leu Leu Glu 225 230 235 240
Leu Ile Ser Tyr Cys Phe Arg Ala Leu Ser Leu Gly Ile Arg Leu Phe 245 250 255Leu Ile Ser Tyr Cys Phe Arg Ala Leu Ser Leu Gly Ile Arg Leu Phe 245 250 255
Ala Asn Met Met Ala Gly His Ser Leu Val Lys Ile Leu Ser Gly Phe 260 265 270Ala Asn Met Met Ala Gly His Ser Leu Val Lys Ile Leu Ser Gly Phe 260 265 270
Ala Trp Thr Met Leu Phe Leu Asn Asn Ile Phe Tyr Phe Ile Gly Asp 275 280 285Ala Trp Thr Met Leu Phe Leu Asn Asn Ile Phe Tyr Phe Ile Gly Asp 275 280 285
Leu Gly Pro Leu Phe Ile Val Leu Ala Leu Thr Gly Leu Glu Leu Gly 290 295 300Leu Gly Pro Leu Phe Ile Val Leu Ala Leu Thr Gly Leu Glu Leu Gly 290 295 300
Val Ala Ile Ser Gin Ala His Val Ser Thr Ile Ser Ile Cys Ile Tyr 305 310 315 320Val Ala Ile Ser Gin Ala His Val Ser Thr Ile Ser Ile Cys Ile Tyr 305 310 315 320
Leu Asn Asp Ala Thr Asn Leu His Gin Asn Glu Ser Phe His Asn 325 330 335Leu Asn Asp Ala Thr Asn Leu His Gin Asn Glu Ser Phe His Asn 325 330 335
(2) INFORMATION POUR LA SEQ ID NO: 5:(2) INFORMATION FOR SEQ ID NO: 5:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 1067 paires de bases(A) LENGTH: 1067 base pairs
(B) TYPE: acide nucléique(B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire
( i i ) TYPE DE MOLECULE : ADNc pour ARN(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA for RNA
( ix ) CARACTERISTIQUE ADDITIONELLE :(ix) ADDITIONAL FEATURE:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT: 99..1067(B) LOCATION: 99..1067
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 5:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC ATTTCGATTT 60GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC ATTTCGATTT 60
TGATGTTGCC ATACAAATAG ATAACAAGCA CAAGCACA ATG CTT TCA CTA CGT 113TGATGTTGCC ATACAAATAG ATAACAAGCA CAAGCACA ATG CTT TCA CTA CGT 113
Met Leu Ser Leu Arg 1 5Met Leu Ser Leu Arg 1 5
CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AGA ACT TTG TGT AGC TCT 161 Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser 10 15 20CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AGA ACT TTG TGT AGC TCT 161 Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser 10 15 20
AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AAA ACT GCC CAA AAC TTA 209 Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys Thr Ala Gin Asn Leu 25 30 35AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AAA ACT GCC CAA AAC TTA 209 Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys Thr Ala Gin Asn Leu 25 30 35
GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GGT CCT GGT GCT AAA GAG 257 Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys Glu 40 45 50GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GGT CCT GGT GCT AAA GAG 257 Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys Glu 40 45 50
GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG ATT CTT CGT TCA TTA TCA 305 Gly Thr Arg Gly Ser Ser Arg Val Glu Met Ile Leu Arg Ser Leu Ser 55 60 65GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG ATT CTT CGT TCA TTA TCA 305 Gly Thr Arg Gly Ser Ser Arg Val Glu Met Ile Leu Arg Ser Leu Ser 55 60 65
TGT CGA TTC TTC ACA ATC GCT CTT TGT GAT GCT GCG GAA CCA TGG CAA 353 Cys Arg Phe Phe Thr Ile Ala Leu Cys Asp Ala Ala Glu Pro Trp Gin 70 75 80 65TGT CGA TTC TTC ACA ATC GCT CTT TGT GAT GCT GCG GAA CCA TGG CAA 353 Cys Arg Phe Phe Thr Ile Ala Leu Cys Asp Ala Ala Glu Pro Trp Gin 70 75 80 65
TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC 401 Leu Gly Ser Gin Asp Ala Ala Thr Pro Met Met Gin Gly Ile Ile Asp 90 95 100TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC 401 Leu Gly Ser Gin Asp Ala Ala Thr Pro Met Met Gin Gly Ile Ile Asp 90 95 100
TTA CAT CAC GAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA 449 Leu His His Asp Ile Phe Phe Phe Leu Ile Leu Ile Leu Val Phe Val 105 110 115TTA CAT CAC GAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA 449 Leu His His Asp Ile Phe Phe Phe Leu Ile Leu Ile Leu Val Phe Val 105 110 115
TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT AAT 497 Ser Arg Met Leu Val Arg Ala Leu Trp His Phe Asn Glu Gin Thr Asn 120 125 130TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT AAT 497 Ser Arg Met Leu Val Arg Ala Leu Trp His Phe Asn Glu Gin Thr Asn 120 125 130
CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATT CGG 545 Pro Ile Pro Gin Arg Ile Val His Gly Thr Thr Met Glu Ile Ile Arg 135 140 145
ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT 593CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATT CGG 545 Pro Ile Pro Gin Arg Ile Val His Gly Thr Thr Met Glu Ile Ile Arg 135 140 145 ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT 593
Thr Ile Phe Pro Ser Val Ile Leu Leu Phe Ile Ala Ile Pro Ser PheThr Ile Phe Pro Ser Val Ile Leu Leu Phe Ile Ala Ile Pro Ser Phe
150 155 160 165150 155 160 165
GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT 641GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT 641
Ala Leu Leu Tyr Ser Met Asp Gly Val Leu Val Asp Pro Ala Ile Thr 170 175 180Ala Leu Leu Tyr Ser Met Asp Gly Val Leu Val Asp Pro Ala Ile Thr 170 175 180
ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC 689ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC 689
Ile Lys Ala Ile Gly His Gin Trp Tyr Arg Thr Tyr Glu Tyr Ser Asp 185 190 195Ile Lys Ala Ile Gly His Gin Trp Tyr Arg Thr Tyr Glu Tyr Ser Asp 185 190 195
TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT 737TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT 737
Tyr Asn Ser Ser Asp Glu Gin Ser Leu Thr Phe Asp Ser Tyr Thr Ile 200 205 210Tyr Asn Ser Ser Asp Glu Gin Ser Leu Thr Phe Asp Ser Tyr Thr Ile 200 205 210
CCA GAA GAT GAT CCA GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC 785CCA GAA GAT GAT CCA GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC 785
Pro Glu Asp Asp Pro Glu Leu Gly Gin Ser Arg Leu Leu Glu Val AspPro Glu Asp Asp Pro Glu Leu Gly Gin Ser Arg Leu Leu Glu Val Asp
215 220 225215 220 225
AAT AGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTA ACA 833AAT AGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTA ACA 833
Asn Arg Val Val Val Pro Ala Lys Thr His Leu Arg Met Ile Val ThrAsn Arg Val Val Val Pro Ala Lys Thr His Leu Arg Met Ile Val Thr
230 235 240 245230 235 240 245
CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA GGT GTC AAA 881CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA GGT GTC AAA 881
Pro Ala Asp Val Pro His Ser Trp Ala Val Pro Ser Ser Gly Val Lys 250 255 260Pro Ala Asp Val Pro His Ser Trp Ala Val Pro Ser Ser Gly Val Lys 250 255 260
TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC TTC ATC TCG GTA CAA 929TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC TTC ATC TCG GTA CAA 929
Cys Asp Ala Val Pro Gly Arg Ser Asn Leu Thr Phe Ile Ser Val Gin 265 270 275Cys Asp Ala Val Pro Gly Arg Ser Asn Leu Thr Phe Ile Ser Val Gin 265 270 275
CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACT AAT 977CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACT AAT 977
Arg Glu Gly Val Tyr Tyr Gly Gin Cys Ser Glu Ile Arg Gly Thr Asn 280 285 290Arg Glu Gly Val Tyr Tyr Gly Gin Cys Ser Glu Ile Arg Gly Thr Asn 280 285 290
CAT GCC TTT ACG CCT ATC GTC GTA GAA GCA GTG ACT TTG AAA GAT TAT 1025CAT GCC TTT ACG CCT ATC GTC GTA GAA GCA GTG ACT TTG AAA GAT TAT 1025
His Ala Phe Thr Pro Ile Val Val Glu Ala Val Thr Leu Lys Asp TyrHis Ala Phe Thr Pro Ile Val Val Glu Ala Val Thr Leu Lys Asp Tyr
295 300 305295,300,305
GCG GAT TGG GTA TCC AAT CAA TTA ATC CTC CAA ACC AAC TAA 1067GCG GAT TGG GTA TCC AAT CAA TTA ATC CTC CAA ACC AAC TAA 1067
Ala Asp Trp Val Ser Asn Gin Leu Ile Leu Gin Thr AsnAla Asp Trp Val Ser Asn Gin Leu Ile Leu Gin Thr Asn
310 315 320310 315 320
(2) INFORMATION POUR LA SEQ ID NO: 6:(2) INFORMATION FOR SEQ ID NO: 6:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 322 acides aminés(A) LENGTH: 322 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: protéine
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 6:(ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Met Leu Ser Leu Arg Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15Met Leu Ser Leu Arg Gin Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15
Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys 20 25 30Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gin Gin Lys Pro Val Val Lys 20 25 30
Thr Ala Gin Asn Leu Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly 35 40 45Thr Ala Gin Asn Leu Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly 35 40 45
Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Ser Arg Val Glu Met Ile 50 55 60Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Ser Arg Val Glu Met Ile 50 55 60
Leu Arg Ser Leu Ser Cys Arg Phe Phe Thr Ile Ala Leu Cys Asp Ala 65 70 75 80Leu Arg Ser Leu Ser Cys Arg Phe Phe Thr Ile Ala Leu Cys Asp Ala 65 70 75 80
Ala Glu Pro Trp Gin Leu Gly Ser Gin Asp Ala Ala Thr Pro Met Met 85 90 95Ala Glu Pro Trp Gin Leu Gly Ser Gin Asp Ala Ala Thr Pro Met Met 85 90 95
Gin Gly Ile Ile Asp Leu His His Asp Ile Phe Phe Phe Leu Ile Leu 100 105 110Gin Gly Ile Ile Asp Leu His His Asp Ile Phe Phe Phe Leu Ile Leu 100 105 110
Ile Leu Val Phe Val Ser Arg Met Leu Val Arg Ala Leu Trp His Phe 115 120 125Ile Leu Val Phe Val Ser Arg Met Leu Val Arg Ala Leu Trp His Phe 115 120 125
Asn Glu Gin Thr Asn Pro Ile Pro Gin Arg Ile Val His Gly Thr Thr 130 135 140Asn Glu Gin Thr Asn Pro Ile Pro Gin Arg Ile Val His Gly Thr Thr 130 135 140
Met Glu Ile Ile Arg Thr Ile Phe Pro Ser Val Ile Leu Leu Phe Ile 145 150 155 160Met Glu Ile Arg Arg Ile Phe Pro Ser Val Ile Leu Leu Phe Ile 145 150 155 160
Ala Ile Pro Ser Phe Ala Leu Leu Tyr Ser Met Asp Gly Val Leu Val 165 170 175Ala Ile Pro Ser Phe Ala Leu Leu Tyr Ser Met Asp Gly Val Leu Val 165 170 175
Asp Pro Ala Ile Thr Ile Lys Ala Ile Gly His Gin Trp Tyr Arg Thr 180 185 190Asp Pro Ala Ile Thr Ile Lys Ala Ile Gly His Gin Trp Tyr Arg Thr 180 185 190
Tyr Glu Tyr Ser Asp Tyr Asn Ser Ser Asp Glu Gin Ser Leu Thr Phe 195 200 205Tyr Glu Tyr Ser Asp Tyr Asn Ser Ser Asp Glu Gin Ser Leu Thr Phe 195 200 205
Asp Ser Tyr Thr Ile Pro Glu Asp Asp Pro Glu Leu Gly Gin Ser Arg 210 215 220Asp Ser Tyr Thr Ile Pro Glu Asp Asp Pro Glu Leu Gly Gin Ser Arg 210 215 220
Leu Leu Glu Val Asp Asn Arg Val Val Val Pro Ala Lys Thr His Leu 225 230 235 240Leu Leu Glu Val Asp Asn Arg Val Val Val Pro Ala Lys Thr His Leu 225 230 235 240
Arg Met Ile Val Thr Pro Ala Asp Val Pro His Ser Trp Ala Val Pro 245 250 255Arg Met Ile Val Thr Pro Ala Asp Val Pro His Ser Trp Ala Val Pro 245 250 255
Ser Ser Gly Val Lys Cys Asp Ala Val Pro Gly Arg Ser Asn Leu Thr 260 265 270
Phe Ile Ser Val Gin Arg Glu Gly Val Tyr Tyr Gly Gin Cys Ser Glu 275 280 285Ser Ser Gly Val Lys Cys Asp Ala Val Pro Gly Arg Ser Asn Leu Thr 260 265 270 Phe Ile Ser Val Gin Arg Glu Gly Val Tyr Tyr Gly Gin Cys Ser Glu 275 280 285
Ile Arg Gly Thr Asn His Ala Phe Thr Pro Ile Val Val Glu Ala Val 290 295 300Ile Arg Gly Thr Asn His Ala Phe Thr Pro Ile Val Val Glu Ala Val 290 295 300
Thr Leu Lys Asp Tyr Ala Asp Trp Val Ser Asn Gin Leu Ile Leu Gin 305 310 315 320Thr Leu Lys Asp Tyr Ala Asp Trp Val Ser Asn Gin Leu Ile Leu Gin 305 310 315 320
Thr Asn
Thr asn
Claims
REVENDICATIONS 1") Planteε tranεgéniqueε, poεεédant danε leurs noyaux, une séquence hybride exprimable (transgène) , comprenant au moins une région codante d'un gène mitochondrial non édité de végétaux supérieurs ' et une séquence susceptible de transférer la protéine expri¬ mée par ladite région codante, vers la mitochondrie, lesquelleε planteε εont caractérisées en ce que :CLAIMS 1") Transgenic plant, containing in their nuclei, an expressible hybrid sequence (transgene), comprising at least one coding region of an unedited mitochondrial gene of higher plants and a sequence capable of transferring the protein expressed by said coding region, towards the mitochondrion, which plant is characterized in that:
- les régions codantes deε gèneε mitochon- driaux non éditéε εont choiεiε parmi leε gèneε codant pour une protéine du complexe ATP εynthaεe choiεiε parmi le fragment de gène de 1ΑTP9 de blé, de formule I εui- vante :- the coding regions of the unedited mitochondrial gene are chosen from the gene coding for a protein of the ATP complex chosen from the wheat 1TP9 gene fragment, of formula I below:
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA ou le gène de 1ΑTP6, ou parmi leε gèneε codant pour une protéine de la chaîne reεpiratoire choiεiε parmi leε gène deε εouε-unitéε 1 à 7 de la NAD déεhydrogénaεe, le gène de 1 'apocytochrome b et leε gènes des εouε-unitéε I, II ou III de la cytochrome-oxydaεe et - la εéquence εuεceptible de tranεférer ladite protéine exprimée verε la mitochondrie eεt sélectionnée danε le groupe conεtitué par leε fragmentε codant pour la tryptophanyl ARNt εynthétaεe de levure, la εouε-unité β de l'ATPaεe de Nicotiana piumbaginifolia , et le tranεlocateur ATP/ADP de mais ou un fragment Ecorl/Kpnl de 303 paireε de baεes incluant les codons 1 à 62 de la souε-unité IV de la cytochrome oxydase de levure, laquelle' séquence hybride est apte à modifier la ferti¬ lité mâle deε planteε ayant incorporé ledit tranεgène, tout en ne modifiant paε ieε autreε caractériεtiqueε phé- notypiqueε deεditeε planteε .
2') Planteε transgéniques selon la revendica¬ tion 1, caractériséeε en ce que ladite εéquence d'acide nucléique hybride comprend la région du gène codant pour la forme non éditée de 1ΑTP9 de blé, de formule εuivante :ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA or the 1ΑTP6 gene, or among the gene coding for a chosen respiratory chain protein among the gene of the unit 1 to 7 of the dehydrogenated NAD, the gene of apocytochrome b and the genes of the unit I, II or III of the cytochrome-oxidation and - the sequence capable of transferring said protein expressed verε the mitochondrion is selected from the group consisting of the fragment coding for the yeast tryptophanyl tRNA synthetium, the β-unit of ATPase from Nicotiana piumbaginifolia, and the ATP/ADP translocator from corn or an Ecorl/Kpnl fragment of 303 pairs of bases including codons 1 to 62 of subunit IV of yeast cytochrome oxidase, which hybrid sequence is capable of modifying the male fertility of the plant having incorporated said tranεgene, while not modifying any other characteristic of the phe - nottypical of the said plant. 2') Transgenic plant according to claim 1, characterized in that said hybrid nucleic acid sequence comprises the region of the gene coding for the unedited form of wheat TP9, of the following formula:
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA, à laquelle eεt associée en tant que séquence de trans¬ fert, les codons 1 à 62 de la pré-séquence de la sous- unité IV de la cytochrome-oxydase (cox IV) de levure (SEQ. ID n" 1) .ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA, with which is associated as a transfer sequence, codons 1 to 62 of the pre-sequence of subunit IV of yeast cytochrome oxidase (cox IV) (SEQ. ID No. 1).
3 ' ) Plantes transgéniqueε selon la revendica¬ tion 1 ou la revendication 2, caractérisées en ce que ladite séquence d'acide nucléique hybride comprend le fragment de région codant pour la forme non éditée de 1ΑTP6 de blé, de formule suivante :3 ') Transgenic plants according to claim 1 or claim 2, characterized in that said hybrid nucleic acid sequence comprises the region fragment coding for the unedited form of wheat 1ΑTP6, of the following formula:
ATG GAT AAT TTT ATC CAG AAT CTG CCT GGT GCC TAC CCG GAAATG GAT AAT TTT ATC CAG AAT CTG CCT GGT GCC TAC CCG GAA
ACC CCA TTG GAT CAA TTT GCC ATT ATC CCA ATA ATT GAT CTTACC CCA TTG GAT CAA TTT GCC ATT ATC CCA ATA ATT GAT CTT
CAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT GAA GTC TTGCAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT GAA GTC TTG
TAT ATG CTG CTC ACT GTC GTT TTG GTC GTT TTT CTT TTT TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCATAT ATG CTG CTC ACT GTC GTT TTG GTC GTT TTT CTT TTT TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCA
TGG CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AACTGG CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC
CTG GTA AAC GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AAACTG GTA AAC GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AAA
CAA AAG TTT TTC CCT CGC ATC TCG GTC ACT TTT ACT TTT TCGCAA AAG TTT TTC CCT CGC ATC TCG GTC ACT TTT ACT TTT TCG
TTA TTT CGT AAT CCC CAG GGT ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG GCT CTT TCA TTT TCCTTA TTT CGT AAT CCC CAG GGT ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG GCT CTT TCA TTT TCC
ATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA AGA CAT GGGATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA AGA CAT GGG
CTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA CTGCTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA CTG
CCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TATCCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TAT
TGT TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT ATG ATG GCC GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTTTGT TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT ATG ATG GCC GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT
GCT TGG ACT ATG CTA TTT CTG AAT AAT ATT TTC TAT TTC ATA
GGA GAT CTT GGT CCC TTA TTT ATA GTT CTA GCA TTA ACC GGT CTG GAA TTA GGT GTA GCT ATA TCA CAA GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA AAT CTC CAT CAA AAT GAG TCA TTT CAT AAT TGA, à laquelle est associée en tant que séquence de trans¬ fert, les codons 1 à 62 de la pré-séquence de la souε- unité IV de la cytochrome-oxydaεe (cox IV) de levure (SEQ. ID n' 3) .GCT TGG ACT ATG CTA TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GAT CTT GGT CCC TTA TTT ATA GTT CTA GCA TTA ACC GGT CTG GAA TTA GGT GTA GCT ATA TCA CAA GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA AAT CTC CAT CAA AAT GAG TCA TTT CAT AAT TGA , with which is associated as a transfer sequence, codons 1 to 62 of the pre-sequence of subunit IV of yeast cytochrome oxidase (cox IV) (SEQ. ID no. 3).
4') Planteε tranεgéniqueε selon l'une quelconque des revendications 1 à 3, caractérisées en ce que ladite séquence d'acide nucléique hybride comprend le fragment de région codant pour la forme non éditée de cox II de formule εuivante : ATG ATT CTT CGT TCA TTA TCA TGT CGA TTC TTC ACA ATC GCT CTT TGT GAT GCT GCG GAA CCA TGG CAA TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC TTA CAT CAC GAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT AAT CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATT CGG ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT CCA GAA GAT GAT CCA GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC AAT4') Tranεgenicε plant according to any one of claims 1 to 3, characterized in that said hybrid nucleic acid sequence comprises the region fragment coding for the unedited form of cox II of the following formula: ATG ATT CTT CGT TCA TTA TCA TGT CGA TTC TTC ACA ATC GCT CTT TGT GAT GCT GCG GAA CCA TGG CAA TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC TTA CAT CAC GAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT AAT CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATT CGG ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT CCA GAA GAT GAT CCA GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC AAT
AGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTA ACA CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA GGT GTC AAA TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC TTC ATC TCG GTA CAA CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACT AAT CAT GCC TTT ACG CCT ATC GTCAGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTA ACA CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA GGT GTC AAA TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC TTC ATC TCG GTA CAA CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACT AAT CAT GCC TTT ACG CCT ATC GTC
GTA GAA GCA GTG ACT TTG AAA GAT TAT GCG GAT TGG GTA TCC AAT CAA TTA ATC CTC CAA ACC AAC TAA, à laquelle eεt aεεociée en tant que εéquence de tranε- fert, leε codons 1 à 62 de la pré-εéquence de la εous- unité IV de la cytochrome-oxydaεe (cox IV) de levure (SEQ. ID n* 5) .
5") Plante tranεgénique selon l'une quelconque des revendications 1 à 4, caractérisée en ce que la plante ayant incorporé ledit transgène eεt du tabac.GTA GAA GCA GTG ACT TTG AAA GAT TAT GCG GAT TGG GTA TCC AAT CAA TTA ATC CTC CAA ACC AAC TAA, to which it is associated as the transfer sequence, the codons 1 to 62 of the pre-sequence of the transfer - unit IV of yeast cytochrome oxidase (cox IV) (SEQ. ID n * 5). 5") Tranεgenic plant according to any one of claims 1 to 4, characterized in that the plant having incorporated said transgene is tobacco.
6*) Plante tranεgénique εelon l'une quelconque deε revendicationε 1 à 5, caractérisée en ce que la plante ayant incorporé ledit transgène est notamment choisie parmi : le colza, le tournesol, le soja, la tomate, la pomme de terre, le melon, la carotte, le piment, l'endive, le trèfle, le lupin, le haricot, le pois, le mais, le blé, le seigle, l'avoine, l'orge, le riz, le millet, leε agrumeε, le coton.6 * ) Tranεgenic plant according to any one of claims 1 to 5, characterized in that the plant having incorporated said transgene is chosen in particular from: rapeseed, sunflower, soya, tomato, potato, melon , carrot, pepper, endive, clover, lupine, bean, pea, corn, wheat, rye, oats, barley, rice, millet, citrus fruit, cotton.
7*) Séquence d'acide nucléique hybride, com¬ prenant au moins la région codante d'un gène mitochon¬ drial non édité de végétaux supérieurε, à laquelle est asεociée une εéquence susceptible de transférer la pro¬ téine exprimée par ladite région codante, vers la mito¬ chondrie, caractérisée en ce que :7 * ) Hybrid nucleic acid sequence, comprising at least the coding region of an unedited mitochondrial gene of higher plants, with which is associated a sequence capable of transferring the protein expressed by said coding region, towards the mitochondria, characterized in that:
- les régions codanteε deε gèneε mitochon¬ driaux non édités sont choisis parmi leε gènes codant pour une protéine du complexe ATP synthaεe choiεiε parmi le fragment de gène de 1 'ATP9 de blé ,de formule I εui- vante :- the coding regions of the unedited mitochondrial gene are chosen from the genes coding for a protein of the ATP synthase complex chosen from the wheat ATP9 gene fragment, of the following formula I:
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACAATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA
ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCTATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT
AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACCAAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC
GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA ou le gène de 1ΑTP6, ou parmi leε gèneε codant pour une protéine de la chaîne respiratoire, choiεiε parmi les gènes des souε-unités 1 à 7 de la NAD déshydrogénase, de 1 'apocytochrome b et des sous-unités I, II ou III de la cytochrome-oxydase, etGAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA or the 1ΑTP6 gene, or from the gene coding for a protein of the respiratory chain, chosen from the genes of the -units 1 to 7 of NAD dehydrogenase, apocytochrome b and subunits I, II or III of cytochrome oxidase, and
- la séquence nucléique susceptible de tranε- férer ladite protéine exprimée verε la mitochondrie eεt sélectionnée danε le groupe conεtitué par deε fragmentε
codant pour la tryptophanyl ARNt εynthétaεe de levure, la εouε-unité β de l'ATPaεe de Nicotiana plu baginifolia , le tranεlocateur ATP/ADP de maiε et un fragment Ecorl/Kpnl de 303 paires de bases incluant les codons 1 à 62 de la εouε-unité IV de la cytochrome oxydase de levure, laquelle séquence hybride est apte à modifier la ferti¬ lité mâle deε plantes l'ayant incorporée.- the nucleic acid sequence capable of transferring said expressed protein to the mitochondrion is selected in the group made up of fragments coding for the tryptophanyl εynthetaε tRNA of yeast, the εouε-β unit of ATPaεe from Nicotiana plu baginifolia, the ATP/ADP tranεlocator of maiε and an Ecorl/Kpnl fragment of 303 base pairs including codons 1 to 62 of the εouε -unit IV of yeast cytochrome oxidase, which hybrid sequence is capable of modifying the male fertility of plants having incorporated it.
8*) Séquence d'acide nucléique hybride selon la revendication 7, caractérisée en ce qu'elle comprend la région du gène codant pour la forme non éditée de 1 'ATP9 de blé, de formule suivante :8 * ) Hybrid nucleic acid sequence according to claim 7, characterized in that it comprises the region of the gene coding for the unedited form of wheat ATP9, of the following formula:
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA, à laquelle est asεociée en tant que séquence de transfert, leε codonε 1 à 62 de la pré-εéquence de la sous-unité IV de la cytochrome-oxydase (cox IV) de levure (SEQ. ID n* 1) .ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA, to which is associated as a transfer sequence, the codon 1 to 62 of the pre-sequence of subunit IV of yeast cytochrome oxidase (cox IV) (SEQ. ID n * 1).
9') Séquence d'acide nucléique hybride selon la revendication 7, caractérisée en ce qu'elle comprend le fragment de région codant pour la forme non éditée de 1 'ATP6 de blé, de formule suivante :9') Hybrid nucleic acid sequence according to claim 7, characterized in that it comprises the region fragment coding for the unedited form of wheat ATP6, of the following formula:
ATG GAT AAT TTT ATC CAG AAT CTG CCT GGT GCC TAC CCG GAA ACC CCA TTG GAT CAA TTT GCC ATT ATC CCA ATA ATT GAT CTT CAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT GAA GTC TTG TAT ATG CTG CTC ACT GTC GTT TTG GTC GTT TTT CTT TTT TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCA TGG CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC CTG GTA AAC GAA CAA ATA GGT ,GGT CTT TCC GGA AAT GTG AAA CAA AAG TTT TTC CCT CGC ATC TCG GTC ACT TTT ACT TTT TCG TTA TTT CGT AAT CCC CAG GGT ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG GCT CTT TCA TTT TCC ATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA AGA CAT GGG
CTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA CTG CCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TAT TGT TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT ATG ATG GCC GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT GCT TGG ACT ATG CTA TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GAT CTT GGT CCC TTA TTT ATA GTT CTA GCA TTA ACC GGT CTG GAA TTA GGT GTA GCT ATA TCA CAA GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA AAT CTC CAT CAA AAT GAG TCA TTT CAT AAT TGA, à laquelle est associée en tant que séquence de trans¬ fert, les codonε 1 à 62 de la pré-εéquence de la sous- unité IV de la cytochrome-oxydase (cox IV) de levure (SEQ. ID n* 3) .ATG GAT AAT TTT ATC CAG AAT CTG CCT GGT GCC TAC CCG GAA ACC CCA TTG GAT CAA TTT GCC ATT ATC CCA ATA ATT GAT CTT CAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT GAA GTC TTG TAT ATG CTG CTC ACT GTC GTT TTG GTC GTT TTT CTT TTT TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCA TGG CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC CTG GTA AAC GAA CAA ATA GGT ,GGT CTT TCC GGA AAT GTG AAA CAA AAG TTT TTC CCT CGC ATC TCG GTC ACT TTT ACT TTT TCG TTA TTT CGT AAT CCC CAG GGT ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG GCT CTT TCA TTT TCC ATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA AGA CAT GGG CTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA CTG CCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TAT TGT TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT ATG ATG GCC GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT GCT TGG ACT ATG CTA TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GAT CTT GGT CCC TTA TTT ATA GTT CTA GCA TTA ACC GGT CTG GAA TTA GGT GTA GCT ATA TCA CAA GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA AAT CTC CAT CAA AAT GAG TCA TTT CAT AAT TGA, with which is associated as a transfer sequence, codons 1 to 62 of the pre-sequence of subunit IV yeast cytochrome oxidase (cox IV) (SEQ. ID No. * 3).
10') Séquence d'acide nucléique hybride selon la revendication 7, caractérisée en ce qu'elle comprend le fragment de région codant pour la forme non éditée de cox II de formule suivante :10') Hybrid nucleic acid sequence according to claim 7, characterized in that it comprises the region fragment coding for the unedited form of cox II of the following formula:
ATG ATT CTT CGT TCA TTA TCA TGT CGA TTC TTC ACA ATC GCT CTT TGT GAT GCT GCG GAA CCA TGG CAA TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC TTA CAT CACATG ATT CTT CGT TCA TTA TCA TGT CGA TTC TTC ACA ATC GCT CTT TGT GAT GCT GCG GAA CCA TGG CAA TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC TTA CAT CAC
GAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT AAT CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATT CGG ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT CCA GAA GAT GAT CCA GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC AAT AGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTAGAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT AAT CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATT CGG ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT CCA GAA GAT GAT CCA GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC AAT AGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTA
ACA CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA GGT GTC AAA TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC TTC ATC TCG GTA CAA CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACT AAT CAT GCC TTT ACG CCT ATC GTC GTA GAA GCA GTG ACT TTG AAA GAT TAT GCG GAT TGG GTA TCC AAT CAA TTA ATC CTC CAA ACC AAC TAA,
à laquelle est asεociée en tant que εéquence de trans¬ fert, les codonε 1 à 62 de la pré-εéquence de la εouε- unité IV de la cytochrome-oxydaεe (cox IV) de levure (SEQ. ID n' 5) . 11") Plaεmide, caractérisé en ce qu'il inclut une séquence d'acide nucléique hybride selon l'une quel¬ conque des revendications 7 à 10, asεociée à un promoteur choisi parmi les promoteurs s 'exprimant constitutivement et les promoteurε ε'exprimant au niveau deε anthèreε et à un terminateur convenable.ACA CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA GGT GTC AAA TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC TTC ATC TCG GTA CAA CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACT AAT CAT GCC TTT ACG CCT ATC GTC GTA GAA GCA GTG ACT TTG AAA GAT TAT GCG GAT TGG GTA TCC AAT CAA TTA ATC CTC CAA ACC AAC TAA, to which is associated as a transfer sequence, codons 1 to 62 of the pre-sequence of the εouε- unit IV of the cytochrome-oxidation (cox IV) of yeast (SEQ. ID no. 5). 11") Plaεmid, characterized in that it includes a hybrid nucleic acid sequence according to any one of claims 7 to 10, associated with a promoter chosen from constitutively expressing promoters and promoters expressing at the level ofε antherε and at a suitable terminator.
12") Plaεmide εelon la revendication 11, ca¬ ractérisé en ce que ladite séquence est sous le contrôle du promoteur 35S et du terminateur du gène VI du CaMV.12") Plaεmid according to claim 11, characterized in that said sequence is under the control of the 35S promoter and the terminator of the CaMV gene VI.
13") Plasmide selon la revendication 11 ou la revendication 12, caractérisé en ce qu'il comprend en outre au moins un gène marqueur.13") Plasmid according to claim 11 or claim 12, characterized in that it further comprises at least one marker gene.
14") Procédé de production de planteε tranε- géniques selon l'une quelconque des revendications 1 à 6, caractérisé en ce qu'il comprend, pour la transformation du végétal supérieur εélectionné, l'introduction d'au moinε une copie de la εéquence nucléique hybride εelon l'une quelconque deε revendicationε 7 à 10, danε une plante réceptrice, à l'aide d'un plasmide contenant la¬ dite εéquence, εelon l'une quelconque deε revendicationε 11 à 13.14") Process for producing transgenic plants according to any one of claims 1 to 6, characterized in that it comprises, for the transformation of the selected higher plant, the introduction of at least one copy of the sequence hybrid nucleic acid according to any one of claims 7 to 10, in a recipient plant, using a plasmid containing the said sequence, according to any one of claims 11 to 13.
15") Procédé selon la revendication 14, carac¬ térisé en ce que ladite tranεformation est obtenue par l'une quelconque des méthodes suivantes : transformation de protoplaεteε, agrotranεformation, microinjection, bio- listique.15") Method according to claim 14, characterized in that said transformation is obtained by any of the following methods: transformation of protoplaεteε, agrotranεformation, microinjection, biology.
16") Procédé d'inhibition de la production de pollen, chez les végétaux supérieurε, caractérisé en ce qu ' il comprend les étapeε εuivanteε :16") Process for inhibiting pollen production in higher plants, characterized in that it comprises the following steps:
(a) inεertion d'une séquence d'acide nucléique hybride, selon l'une quelconque de revendications 7 à 10, chez les végétaux sélectionnéε, par tout moyen appro-
prié ;(a) insertion of a hybrid nucleic acid sequence, according to any one of claims 7 to 10, into the selected plants, by any appropriate means prayed;
(b) régénération et culture des végétaux transgéniqueε obtenuε en (a) ; et(b) regeneration and cultivation of the transgenic plants obtained in (a); And
(c) meεure de la production et de la viabilité du pollen (teεt de germination, notamment) .(c) measurement of pollen production and viability (germination time, in particular).
17") Procédé de reεtauration de planteε mâle- fertiles, à partir de plantes tranεgéniques mâle-stérile, selon l'une quelconque des revendications 1 à 6, caracté¬ risé en ce qu'il comprend les étapeε suivantes : (1) transformation du végétal supérieur sélec¬ tionné par l'introduction d'au moins une copie de la sé¬ quence nucléique hybride selon l'une quelconque des re¬ vendications 7 à 10, dans une plante réceptrice, à l'aide d'un plasmide contenant ladite séquence, pour l'obtention de plantes transgéniques mâle-stériles (PTMS) ;17") Process for restoring male-fertile plants, from male-sterile transgenic plants, according to any one of claims 1 to 6, characterized in that it comprises the following steps: (1) transformation of the higher plant selected by the introduction of at least one copy of the hybrid nucleic sequence according to any one of claims 7 to 10, into a recipient plant, using a plasmid containing said sequence, for obtaining transgenic male-sterile plants (PTMS);
(2) transformation du même végétal supérieur qu'en (1), par l'introduction d'au moins une copie d'une séquence nucléique hybride anti-senε, incluant au moins la même région codante de gène mitochondrial non édité de végétaux que celle incluse dans lesdites planteε tranε- géniqueε mâle-stérile obtenues en (1) , dans une plante réceptrice, à l'aide d'un plasmide contenant ladite sé¬ quence, pour l'obtention de plantes transgéniques mâle- fertiles (PTMF) ; (3) croisement deε planteε tranεgéniques mâle- εtérileε obtenues en (1) et des plantes mâle-fertiles ob- tenueε en (2), pour l'obtention d'hybrideε.(2) transformation of the same higher plant as in (1), by the introduction of at least one copy of an anti-senε hybrid nucleic sequence, including at least the same coding region of unedited mitochondrial gene of plants as that included in said male-sterile transgenic plant obtained in (1), in a recipient plant, using a plasmid containing said sequence, for obtaining male-fertile transgenic plants (PTMF); (3) crossing of male-sterile transgenic plants obtained in (1) and male-fertile plants obtained in (2), to obtain hybrids.
18") Planteε transgéniques, caractérisées en ce qu'elles comprennent dans leurs noyaux une εéquence d'acide nucléique hybride dite anti-εens, c'est-à-dire incluant au moins la même région codante de gène mito¬ chondrial non édité de végétaux que celle incluse dans les plantes transgéniqueε mâle-εtérile εelon l'une quel¬ conque deε revendicationε 1 à 6, danε le εenε inverεe, leεquelleε planteε tranεgéniqueε sont aptes à restaurer les plantes transgéniqueε εelon l'une quelconque deε
revendications 1 à 6.18") Transgenic plants, characterized in that they comprise in their nuclei a hybrid nucleic acid sequence called anti-εens, that is to say including at least the same coding region of unedited mitochondrial gene of plants than that included in male-sterile transgenic plants according to any of claims 1 to 6, in the reverse case, which transgenic plants are capable of restoring transgenic plants according to any of claims 1 to 6.
19") Plaεmide, caractériεé en ce qu'il inclut une région codante d'un gène mitochondrial non édité de végétaux anti-sens, associée à un promoteur choisi parmi les promoteurs s 'exprimant constitutivement et les promo¬ teurs s 'exprimant au niveau deε anthères et à un termina¬ teur convenable.
19") Plaεmid, characterized in that it includes a coding region of an unedited mitochondrial gene of antisense plants, associated with a promoter chosen from the promoters expressing constitutively and the promoters expressing at the level deε anthers and a suitable terminator.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9301650A FR2703561B1 (en) | 1993-02-15 | 1993-02-15 | TRANSGENIC PLANTS INCLUDING A TRANSGEN CONSISTING OF A HYBRID NUCLEIC ACID SEQUENCE, COMPRISING AT LEAST ONE FRAGMENT OF UNMITTED MITOCHONDRIAL GENE OF SUPERIOR PLANTS AND PREPARATION. |
FR9301650 | 1993-02-15 | ||
PCT/FR1994/000162 WO1994018334A1 (en) | 1993-02-15 | 1994-02-15 | Transgenic plants including a transgene consisting of a hybrid nucleic acid sequence, comprising at least one non-edited mitochondrial gene fragment of superior plants and process for their production |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0683819A1 true EP0683819A1 (en) | 1995-11-29 |
Family
ID=9444044
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP94906945A Withdrawn EP0683819A1 (en) | 1993-02-15 | 1994-02-15 | Transgenic plants including a transgene consisting of a hybrid nucleic acid sequence, comprising at least one non-edited mitochondrial gene fragment of superior plants and process for their production |
Country Status (5)
Country | Link |
---|---|
US (2) | US5914447A (en) |
EP (1) | EP0683819A1 (en) |
JP (1) | JPH08506245A (en) |
FR (1) | FR2703561B1 (en) |
WO (1) | WO1994018334A1 (en) |
Families Citing this family (16)
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US5549859A (en) * | 1992-08-11 | 1996-08-27 | E. Khashoggi Industries | Methods for the extrusion of novel, highly plastic and moldable hydraulically settable compositions |
FR2703561B1 (en) * | 1993-02-15 | 1995-08-04 | Centre Nat Rech Scient | TRANSGENIC PLANTS INCLUDING A TRANSGEN CONSISTING OF A HYBRID NUCLEIC ACID SEQUENCE, COMPRISING AT LEAST ONE FRAGMENT OF UNMITTED MITOCHONDRIAL GENE OF SUPERIOR PLANTS AND PREPARATION. |
AUPN225695A0 (en) * | 1995-04-07 | 1995-05-04 | Australian National University, The | Plants with altered mitochondrial function |
FR2749321B1 (en) * | 1996-05-31 | 1998-08-21 | Florimond Desprez Veuve Et Fil | RECOMBINANT PLANT GENOME, MITOCHONDRIA AND CELL CONTAINING THE SAME, AND METHOD FOR SELECTING MALE CYTOPLASMIC STERILITY IN A CICHORIUM PLANT |
US6852908B2 (en) | 1997-05-30 | 2005-02-08 | Mcgill University | Method for enhancement of naturally occurring cytoplasmic male sterility and for restoration of male fertility and uses thereof in hybrid crop production |
EP0983371A1 (en) * | 1997-05-30 | 2000-03-08 | THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING (McGILL UNIVERSITY) | Method for enhancement of naturally occurring cytoplasmic male sterility and for restoration of male fertility and uses thereof in hybrid crop production |
US7696416B2 (en) * | 1999-05-04 | 2010-04-13 | Marlin Edwards | Non-pungent ornamental peppers |
EP1261730A2 (en) * | 2000-02-25 | 2002-12-04 | Avesta Gengraine Technologies Pvt. Ltd | A process for generating cytoplasmic male sterile line in rice and other crops by rna editing |
EP1404814B1 (en) * | 2001-07-12 | 2013-06-19 | McGill University | Nuclear fertility restorer genes and methods of use in plants |
US7314971B2 (en) * | 2001-07-12 | 2008-01-01 | Basf Plant Science Gmbh | Nuclear fertility restorer genes and methods of use in plants |
CA2460872A1 (en) * | 2001-09-19 | 2003-04-03 | Japan Tobacco Inc. | A method for providing and inhibiting the rice fertility, and discerning the presence of the rice restorer gene by using the rice restorer gene to the rice bt type cytoplasmic male sterility |
US20060148003A1 (en) * | 2002-08-13 | 2006-07-06 | Campbell Douglas A | Peptide sequence tags and method of using same |
KR20030045732A (en) * | 2003-05-09 | 2003-06-11 | 재단법인서울대학교산학협력재단 | Expression of the CMS-associated gene in pepper for transgenic male sterile plant |
FR2935987B1 (en) * | 2008-09-16 | 2013-04-19 | Centre Nat Rech Scient | IMPORTATION OF A RIBOZYME IN VEGETABLE MITOCHONDRIES BY AN AMINOACYLABLE PSEUDO-ARNTA BY VALINE |
CN107177600B (en) * | 2017-06-29 | 2020-07-07 | 中国水稻研究所 | Rice male sterility gene OsFINGL 1 and application thereof |
CN113430209B (en) * | 2020-03-05 | 2022-09-20 | 山东农业大学 | Barley male sterility gene BMS-1 and application thereof |
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NZ227835A (en) * | 1988-02-03 | 1992-09-25 | Paladin Hybrids Inc | Antisense gene systems of pollination control for hybrid seed production |
WO1990008828A2 (en) * | 1989-02-02 | 1990-08-09 | Paladin Hybrids Inc. | Molecular methods of hybrid seed production |
ATE496135T1 (en) * | 1989-08-10 | 2011-02-15 | Bayer Bioscience Nv | PLANTS WITH MODIFIED FLOWERS |
FR2703561B1 (en) * | 1993-02-15 | 1995-08-04 | Centre Nat Rech Scient | TRANSGENIC PLANTS INCLUDING A TRANSGEN CONSISTING OF A HYBRID NUCLEIC ACID SEQUENCE, COMPRISING AT LEAST ONE FRAGMENT OF UNMITTED MITOCHONDRIAL GENE OF SUPERIOR PLANTS AND PREPARATION. |
-
1993
- 1993-02-15 FR FR9301650A patent/FR2703561B1/en not_active Expired - Fee Related
-
1994
- 1994-02-15 JP JP6517744A patent/JPH08506245A/en active Pending
- 1994-02-15 US US08/505,218 patent/US5914447A/en not_active Expired - Fee Related
- 1994-02-15 EP EP94906945A patent/EP0683819A1/en not_active Withdrawn
- 1994-02-15 WO PCT/FR1994/000162 patent/WO1994018334A1/en not_active Application Discontinuation
-
1999
- 1999-01-04 US US09/224,997 patent/US6479735B1/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
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See references of WO9418334A1 * |
Also Published As
Publication number | Publication date |
---|---|
US6479735B1 (en) | 2002-11-12 |
WO1994018334A1 (en) | 1994-08-18 |
FR2703561B1 (en) | 1995-08-04 |
US5914447A (en) | 1999-06-22 |
FR2703561A1 (en) | 1994-10-14 |
JPH08506245A (en) | 1996-07-09 |
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