Classifications

• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

• the present invention relates to a new plant gene SGS3 and its use for the preparation of genetically modified plants.
• heterologous gene expression will depend on various factors, including the locus of integration of the heterologous gene in the genome of the transformed plant and so-called phenomena of "silencing". It is indeed known to the state of the art as the expression of a heterologous gene in a plant can be inhibited completely or partly in the progeny of the transformed plants regenerated, even said the gene s is expressed correctly in the regenerated plant coming directly from the transformed cell.
• the introduced heterologous gene can sometimes undergo epigenetic inactivation (inactivation being accompanied by any change in sequence). When the genes have homologies with genes of the host organism, the inactivation can also affect the expression of these host genes and generate deleterious effects for the organism (co-inactivation). Two distinct mechanisms of inactivation have been identified in higher plants, translating to either a transcription blocking (transcriptional inactivation) or by RNA degradation (post-transcriptional inactivation).
• GUSs under the control of the CamV 35S promoter showed inactivation of the transgene regardless of the number of copies of the transgene inserted at the locus.
• the phenomenon takes place during the development of each generation indicating a meiotic reversibility.
• Haploid plants from the anther culture of inactivated homozygous transformants carrying a single copy of the transgene showed gene reactivation followed by inactivation during development, suggesting that meiosis was necessary to initiate the reactivation process, but that initiation of inactivation during development did not require fertilization, and was not the result of interaction between different copies of the transgene.
• run-on experiments have shown that the phenomenon occurs at the post-transcriptional level (Elmayan and Vaucheret, Plant J. 9: 787-797,1996).
• SGS3 a new plant gene, called SGS3, involved in the phenomena of post-transcriptional inactivation in transgenic plants and in the resistance of plants to viral infections.
• the inhibition of this gene leads to the inhibition of post-transcriptional inactivation phenomena in particular in transgenic plants comprising a heterologous gene coding for a particular peptide or protein, allowing a level of expression of said peptide or said protein at a particularly high level.
• Another subject of the invention is the overexpression of the SGS3 gene for the preparation of plants more resistant to viral infections.
• SEQ ID NO.l SGS3 gene from Arabidopsis thaliana
• SEQ ID NO. 2 cDNA of the SGS3 gene to Arabidopsis thaliana
• the present invention relates to SGS3 polynucleotides, in particular polynucleotides comprising a plant SGS3 gene.
• the polynucleotides of the present invention comprise the coding sequence of a plant SGS3 gene.
• the SGS3 gene can be isolated in dicotyledonous plants, such as Arabidopsis, tobacco, rapeseed, sunflower, soybean, cotton, clover, duckweed (lemnae) or in monocotyledonous plants such as rice, corn or wheat.
• the SGS3 gene is isolated from dicotyledonous plants, in particular crucifers like Arabidopsis or rapeseed.
• the polynucleotides of the invention comprise an SGS3 d! Arabidopsis thaliana.
• SGS3 polynucleotides designates all of the polynucleotides of the present invention, preferably the polynucleotides of the genomic sequence of SGS3, the polynucleotides of the cDNA sequence of SGS3. as well as the polynucleotides encoding the SGS3 polypeptides of the present invention.
• SGS3 polynucleotides also denotes recombinant polynucleotides comprising the said polynucleotides.
• polynucleotide means a single-stranded nucleotide chain or its complement or a double-stranded nucleotide chain which may be of DNA or RNA type.
• the polynucleotides of the invention are of the DNA type, in particular double stranded DNA.
• polynucleotide also denotes modified oligonucleotides and polynucleotides.
• polynucleotides of the present invention are isolated or purified from their natural environment.
• the polynucleotides of the present invention can be prepared by conventional techniques of molecular biology as described by Sambrook et al. (Molecular Cloning: A Labratory Manual. 1989) or by chemical synthesis.
• the invention includes polynucleotides of the genomic sequence of the SGS3 gene.
• This genomic sequence includes 5 exons (positions 696-1658, 1732-2023, 2135-2379, 2482-2648, 2739-2949 of SEQ ID NO. 1), 4 introns (positions 1659-1731, 2024-2134, 2380- 2481, 2649-2738 of SEQ ID NO.l) and regulatory sequences in 5 'and 3'.
• the polynucleotides of the genomic sequence of SGS3 comprise a polynucleotide chosen from the following polynucleotides: a) the polynucleotide of SEQ ID NO.1, b) a polynucleotide comprising at least one exon of SEQ ID NO.1; c) a polynucleotide comprising a combination of exons of SEQ ID NO.1.
• the present invention also relates to a polynucleotide comprising a regulatory sequence 5 ′ or 3 ′ of the SGS3 gene.
• the invention relates to a 5 'regulatory polynucleotide comprising the polynucleotide whose sequence is between position 1 and position 695 of SEQ ID NO. 1.
• the invention relates to a 3 'regulatory polynucleotide comprising the polynucleotide whose sequence is between position 2950 and position 3275 of SEQ ID NO. 1.
• the invention also relates to a promoter of the SGS3 gene of Arabidopsis thaliana.
• the promoter of the SGS3 gene comprises a polynucleotide whose sequence is between position 1 and position 695 of SEQ ID NO. 1.
• the promoter of the SGS3 gene comprises a fragment biologically active of a polynucleotide whose sequence is between position 1 and position 695 of SEQ ID NO. 1.
• biologically active fragment is meant above a polynucleotide having a promoter activity and preferably a promoter activity in plants.
• the techniques making it possible to establish the promoter activity of a polynucleotide are well known to those skilled in the art. These techniques conventionally involve the use of an expression vector comprising, in the direction of transcription, the polynucleotide to be tested and a reporter gene (see Sambrook et al., Molecular Cloning: A Labratory Manual. 1989).
• polynucleotides comprising a polynucleotide chosen from the following polynucleotides: a) a polynucleotide homologous to a polynucleotide whose sequence is between position 1 and position 695 of SEQ ID NO. 1, b) a polynucleotide capable of s '' selectively hybridize to a polynucleotide whose sequence is between position 1 and position 695 of SEQ ID NO 1.
• these polynucleotides have promoter activity in plant cells and plants.
• the invention also relates to a terminator sequence of the SGS3 gene of Arabidopsis thaliana.
• the terminator sequence of the SGS3 gene comprises a polynucleotide whose sequence is between position 2950 and position 3275 of SEQ ID NO. 1.
• the terminator sequence of the SGS3 gene comprises a biologically active fragment of a polynucleotide whose sequence is between position 2950 and position 3275 of SEQ ID NO. 1.
• the invention also relates to polynucleotides of the SGS3 cDNA.
• the polynucleotides of the coding sequence of a plant SGS3 gene comprise polynucleotides of SEQ ID NO. 2.
• the invention also extends to polynucleotides comprising a polynucleotide chosen from the following polynucleotides: a) a polynucleotide homologous to a polynucleotide according to SEQ ID No. 1 or SEQ ID No. 2; b) a polynucleotide capable of hybridizing selectively to a polynucleotide according to SEQ ID No. 1 or SEQ ID No. 2.
• the polynucleotides which are homologous to a reference polynucleotide or which selectively hybridize to a reference polynucleotide retain the function of the reference sequence.
• the polynucleotides of the present invention preferably code for a polypeptide essential for post-transcriptional inactivation in plants.
• the polynucleotides of the present invention restore a sgs3 mutant of Arabidopsis thaliana. These mutants and their method of production are described in Elmayan et al. (Plant Cell, 10: 1747-1757, 1998). Other methods for constructing Arabidopsis thaliana mutants in which the SGS3 gene is inactive are well known to those skilled in the art. The methods for obtaining mutants of Arabidopsis thaliana are widely described in the literature.
• homologous a polynucleotide having one or more sequence modifications compared to the reference sequence. These modifications can be deletions, additions or substitutions of one or more nucleotides of the reference sequence.
• the percentage of homology will be at least 70%, 75%, 80%, 85%, 90%, 95% and preferably at least 98% and more preferably at least 99% by compared to the reference sequence.
• the methods for measuring and identifying the homologies between the nucleic acid sequences are well known to those skilled in the art. One can use for example the PILEUP or BLAST programs (in particular Altschul et al., J.
• the invention therefore relates to polynucleotides comprising polynucleotides having at least 70%, 75%, 80%. 85%, 90%, 95%, 98% and preferably at least 98% and more preferably at least 99% homology with the polynucleotides SGS3, the polynucleotides of SEQ ED NO.l or the polynucleotides of SEQ ID NO . 2.
• the invention relates to a polynucleotide comprising a polynucleotide of at least 50, 100, 200, 300, 400, 500, 1000 nucleotides having at least 70%, 75%, 80%, 85%, 90%, 95 %, 98% and preferably at least 98% and more preferably at least 99% homology with the polynucleotides SGS3, the polynucleotides of SEQ ID NO.l or the polynucleotides of SEQ ID NO. 2.
• these homologs retain the function of the reference sequence.
• sequence capable of selective hybridization is meant according to the invention the sequences which hybridize with the reference sequence at a level above the background noise significantly.
• the level of the signal generated by the interaction between the sequence capable of selective hybridization and the reference sequences is generally 10 times, preferably 100 times more intense than that of the interaction of the other DNA sequences generating the background noise.
• the stringent hybridization conditions allowing selective hybridization are well known to those skilled in the art. In general, the hybridization and washing temperature is at least 5 ° C. lower than the Tm of the reference sequence at a given pH and for a given ionic strength.
• the hybridization temperature is at least 30 ° C for a polynucleotide of 15 to 50 nucleotides and at least 60 ° C for a polynucleotide of more than 50 nucleotides.
• the hybridization is carried out in the following buffer: 6X SSC. 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, 500 ⁇ g / ml denatured salmon sperm DNA.
• the washes are for example carried out successively at low stringency in a 2X SSC buffer, 0.1% SDS, at medium stringency in a 0.5X SSC buffer, 01% SDS and at high stringency in a 0.1X SSC buffer, 0.1 % SDS.
• the hybridization can of course be carried out according to other usual methods well known to those skilled in the art (see in particular Sambrook et al., Molecular Cloning: A Labratory Manual, 1989).
• the invention therefore relates to polynucleotides comprising a polynucleotide capable of hybridizing selectively with the polynucleotide of SEQ ID NO.l or the polynucleotide of SEQ ID NO. 2.
• the invention relates to a polynucleotide comprising a polynucleotide of at least 50, 100, 200, 300, 400, 500, 1000 nucleotides capable of hybridizing selectively with the polynucleotide of SEQ ID NO.l or the polynucleotide of SEQ ID NO. 2.
• these polynucleotides which hybridize selectively to a reference polynucleotide retain the function of the reference sequence.
• the present invention also relates to antisense polynucleotides allowing the inhibition of the expression of a plant SGS3 gene.
• the antisense polynucleotides hybridize specifically to the mRNA of a plant SGS3 gene thus interfering with the expression of this gene.
• Techniques for inhibiting the expression of a protein by an antisense polynucleotide are well known to those skilled in the art and widely described in the literature, in particular by Judelson et al. (Gene, 133: 63-69, 1993) as well as by Prokish et al. (Mol.Gen.Genet. 256: 104-114, 1997).
• the antisense polynucleotides of the present invention hybridize to the mRNA of a plant SGS3 gene over its entire length or only to part of the mRNA of a plant SGS3 gene.
• the antisense polynucleotides of the present invention can be perfectly complementary to the mRNA of a plant SGS3 gene or sufficiently homologous to allow pairing and inhibition of the expression of a plant SGS3 gene.
• the present invention therefore also relates to polynucleotides comprising an antisense polynucleotide of a plant SGS3 gene and preferably an antisense polynucleotide of the coding sequence of the SGS3 gene of SEQ ID NO. 2.
• the antisense polynucleotides of the present invention are derived from a polynucleotide of SEQ ID NO. 2.
• the antisense polynucleotides of the present invention comprise the polynucleotide of SEQ ID No.2.
• the antisense polynucleotides of the present invention comprise a fragment of at least 100 nucleotides, preferably of at least 500 nucleotides and preferably of at least 1000 nucleotides of SEQ ID NO.2.
• the antisense polynucleotides of the present invention comprise a polynucleotide having at least 85%, 90%), 95% and preferably at least 98% and more preferably at least 99% homology with a polynucleotide of SEQ ID NO. 2.
• the antisense polynucleotides of the present invention comprise a polynucleotide having at least 85%, 90%, 95% and preferably at least 98% and more preferably at least 99% of homology with a fragment d '' at least 100 nucleotides, preferably at least 500 nucleotides and preferably at least 1000 nucleotides of SEQ ID NO.2.
• the antisense polynucleotides of the present invention specifically inhibit the expression of an SGS3 gene in plants.
• the antisense polynucleotide of the present invention are expressed in plant cells or plants from an expression cassette.
• the present invention relates to the use of a polynucleotide or of a fragment of a polynucleotide of SEQ ID NO. 1 and SEQ ID NO. 2 according to the invention for the identification of the SGS3 gene in other plants.
• Cloning is carried out, for example, by screening cDNA libraries or genomic DNA libraries with a polynucleotide or a fragment of a polynucleotide of SEQ ID NO.l and SEQ ID NO.2. These libraries can also be screened by PCR using specific or degenerate oligonucleotides derived from SEQ ID NO.l or SEQ ID NO.2.
• Plant SGS3 genes can also be identified in databases by nucleotide or protein BLAST using SEQ ID NOs. 1-3. Preferably, it is verified that the cloned genes perform the same function as the gene
• a subject of the invention is also polynucleotides comprising a polynucleotide encoding a polypeptide according to the invention.
• the present invention also relates to SGS3 polypeptides.
• SGS3 polypeptides designates all of the polypeptides of the present invention as well as the polypeptides for which the polynucleotides of the present invention encode.
• SGS3 polypeptides also refers to fusion proteins, recombinant proteins or chimeric proteins comprising these polypeptides.
• polypeptide also denotes proteins and peptides as well as modified polypeptides.
• the polypeptides of the invention are isolated or purified from their natural environment.
• the polypeptides can be prepared by various methods. These methods include purification from natural sources such as cells naturally expressing these polypeptides, production of recombinant polypeptides by appropriate host cells and their subsequent purification, production by chemical synthesis or, finally, a combination of these different approaches. . These various production methods are well known to those skilled in the art.
• the SGS3 polypeptides of the present invention can be isolated from plants expressing SGS3 polypeptides.
• the SGS3 polypeptides of the present invention are isolated from recombinant host organisms expressing a heterologous SGS3 polypeptide or expressing a natural SGS3 polypeptide under the control of a heterologous promoter.
• These organizations are preferably chosen from bacteria, yeasts, fungi, animal cells, plant cells or plants.
• the subject of the present invention is a polypeptide of sequence SEQ ID NO.3 as well as a polypeptide comprising a polypeptide of sequence SEQ ID NO. 3.
• the invention also comprises polypeptides comprising a fragment or a homolog of a SGS3 polypeptide and more particularly of the polypeptide of SEQ ID NO. 3.
• fragment of a polypeptide refers to a polypeptide comprising part but not all of the polypeptide from which it is derived.
• the invention relates to a polypeptide comprising a fragment of at least 10, 15, 20, 25, 30, 35, 40, 50 amino acids of a polypeptide of SEQ ID NO.3.
• these fragments retain at least one biological activity of the polypeptide from which they are derived.
• this activity relates to post-transcriptional inactivation in plants.
• the polypeptides of the present invention restore a sgs3 mutant of Arabidopsis thaliana.
• homologous denotes a polypeptide according to the invention denotes a polypeptide which may have a deletion, an addition or a substitution of at least one amino acid.
• the subject of the invention is a polypeptide having at least 75%, 80%, 85%, 90% or 95%), 98%> and preferably 99% "of identical amino acids with a polypeptide of SEQ ID NO. 3.
• these homologous polypeptides retain the same biological activity.
• this activity relates to post-transcriptional inactivation in plants.
• the polypeptides of the present invention restore a sgs3 mutant of Arabidopsis thaliana.
• the SGS3 gene can be expressed or over-expressed in different host organisms such as plants.
• the present invention relates in particular to the overexpression of the SGS3 gene in plants or plant cells to improve their resistance to viruses.
• the SGS3 gene can be expressed in a host organism under the control of the SGS3 promoter of the present invention or under the control of a heterologous promoter and preferably under the control of a promoter functional in plants.
• a polynucleotide encoding an SGS3 polypeptide is inserted into an expression cassette using cloning techniques well known to those skilled in the art. This expression cassette includes the elements necessary for transcription and translation of the sequences coding for the SGS3 polypeptide.
• this expression cassette comprises both elements making it possible to have an SGS 3 polypeptide produced by a host cell and elements necessary for the regulation of this expression.
• the expression cassettes according to the invention comprise, in the direction of transcription, a functional promoter in a host organism, a plant SGS3 gene or the coding sequence of a plant SGS3 gene and a terminator sequence in said host organism.
• the expression cassettes according to the invention comprise, in the sense of transcription, a functional promoter in a host organism, a polynucleotide encoding an SGS3 polypeptide and a terminator sequence in said host organism.
• the expression cassette comprises, in the direction of transcription, a promoter functional in a host organism, a polynucleotide chosen from the following polynucleotides: a) a polynucleotides encoding a polypeptide SGS3 of SEQ ID NO. 3, for a homolog or for a fragment of a polypeptide of SEQ ID NO.3; b) a polynucleotide of SEQ ID NO. 1; c) a polynucleotide of SEQ ID NO.
• the expression cassettes of the present invention allow the expression of an antisense polynucleotide for the inhibition of expression of the SGS3 gene in a plant.
• the expression cassettes according to the invention comprise, in the direction of transcription, a promoter functional in a host organism, an antisense polynucleotide of the coding sequence of a SGS3 gene. of a plant and a functional terminator sequence in said host organism.
• the expression cassettes according to the invention comprise, in the direction of transcription, a promoter functional in a host organism, an antisense polynucleotide of the coding sequence of the SGS3 gene of SEQ ID NO. 2 and a functional terminator sequence in said host organism.
• the antisense polynucleotides of the present invention are expressed under the control of an inducible promoter.
• the subject of the invention is an expression cassette comprising, in the direction of transcription: a) a promoter functional in a host organism; and b) an SGS3 polynucleotide according to the invention in antisense orientation; and c) a terminator sequence in said host organism.
• the SGS3 promoter can be used to express a heterologous gene in a host organism and in particular in plant cells or in plants.
• the invention therefore also relates to expression cassettes comprising the promoter of a plant SGS3 gene operatively associated with a sequence coding for a heterologous protein, allowing the expression of said protein in plant cells or plants.
• the expression cassette according to the invention comprises, in the direction of transcription, the SGS3 promoter from Arabidopsis thaliana. the coding sequence for the heterologous protein and a functional terminator sequence in the plant cells and plants.
• the expression cassette according to the invention comprises, in the direction of transcription, a polynucleotide whose sequence is between position 1 and position 695 of SEQ ID NO. 1 or a biologically active fragment of the polynucleotide whose sequence is between position 1 and position 695 of SEQ ID NO. 1, the sequence coding for a heterologous polypeptide and a terminator sequence functional in plant cells and plants.
• the expression cassettes according to the present invention can also include any other sequence necessary for the expression of the gene of interest, such as for example regulatory elements or signal sequences allowing the addressing of the polypeptide of interest.
• the present invention also relates to a polynucleotide comprising an expression cassette according to the invention and in particular a vector comprising an expression cassette according to the invention.
• the expression cassettes according to the present invention are inserted into a vector for their replication or for the transformation of a host organism.
• Certain elements of the expression cassettes according to the invention are illustrated below without implied limitation. Promoters
• any type of promoter sequence can be used in the expression cassettes according to the invention.
• the choice of promoter will depend in particular on the host organism chosen for the expression of the gene of interest.
• the present invention relates more particularly to the transformation of plants.
• the choice of promoter used in the expression cassette determines the temporal and spatial expression of the gene of interest. Certain promoters allow specific expression in certain tissues of the plant (roots, leaves or seeds for example) or in certain cells of the plant. Some promoters allow constitutive expression while other promoters are on the contrary inducible.
• promoter regulatory sequence in plants any promoter sequence of a gene expressing itself naturally in plants can be used, in particular a promoter expressing in particular in the leaves of plants, such as for example so-called promoters of original origin.
• bacterial, viral or vegetable or also so-called light-dependent promoters such as that of a gene for the small ribulose-biscarboxylase / oxygenase (RuBisCO) subunit of a plant or any suitable known promoter which can be used.
• RuBisCO small ribulose-biscarboxylase / oxygenase
• any suitable known promoter which can be used.
• promoters of plant origin mention will be made of the histone promoters as described in application EP 0 507 698, or the rice actin promoter (US Pat. No. 5,641,876).
• the promoters of a plant virus gene there may be mentioned that of the cauliflower mosaic (CAMV 19S or 35S), or the circovirus promoter (AU 689 311).
• a promoter regulatory sequence specific to specific regions or tissues of plants and more particularly specific promoters of seeds (Datla et al., Biotechnology Ann. Rev. 3: 269-296, 1997). It is also possible to use an inducible promoter advantageously chosen from the promoters of phenylalanine ammonia lyase (PAL), of HMG-CoA reductase (HMG). of chitinases. glucanases.
• PAL phenylalanine ammonia lyase
• HMG HMG-CoA reductase
• chitinases glucanases.
• PI proteinase
• PR1 family genes nopaline synthase (nos) or the vspB gene (US 5,670,349)
• HMG2 promoter US 5,670,349
• beta promoter apple galactosidase ABS 5,670,349
• ACC synthase ACC synthase
• any regulatory sequence can be used which makes it possible to increase the level of expression of the coding sequence inserted in said expression cassette.
• transcription activators e.g., transcription activators
• leader sequences derived from viruses mention will be made, for example, of the activator of the tobacco mosaic virus (TMV) described in application WO 87/07644, or the activator of the tobacco etch virus (TEV).
• TMV tobacco mosaic virus
• TMV tobacco mosaic virus
• TMV tobacco mosaic virus
• TMV tobacco mosaic virus
• TMV tobacco etch virus
• Different sequences derived from plant introns can also be used to increase the level of expression of the gene of interest, especially in monocotyledonous plants.
• Terminator Sequences A wide variety of terminator sequences can be used in the expression cassettes according to the invention. These sequences allow the termination of transcription and polyadenylation of the mRNA. Any terminator sequence functional in the selected host organism can be used. For expression in plants, it is possible in particular to use the terminator nos of Agrobacterium tumefaciens, or alternatively terminator sequences of plant origin, such as for example the histone terminator (see EP 0 633 317), the terminator CaMV 35 S and the tml terminator. These terminator sequences are usable in monocotyledonous and dicotyledonous plants.
• the terminator sequence of the SGS3 gene from Arabidopsis thaliana is another example of a terminator sequence which can be used in the expression cassettes according to the invention.
• any gene of interest can be expressed in a host organism under the control of an SGS3 promoter.
• the SGS3 promoter is used for the expression of a heterologous gene in plant cells or in a plant.
• the genes of interest which may be expressed in plants under the control of an SGS3 promoter are more broadly illustrated below.
• the present invention also relates to transformation or expression vectors comprising at least one SGS3 polynucleotide or an expression cassette according to the present invention.
• the vectors of the present invention are used in particular for transforming a host organism and for expressing an SGS3 polypeptide or an SGS3 polynucleotide in said host organism.
• the host organism is for example a bacterium, a yeast, a fungus, a plant cell or a plant.
• This vector can in particular consist of a plasmid, a cosmid, a bacteriophage or a virus into which is inserted a polynucleotide SGS3 or an expression cassette according to the invention.
• any vector capable of maintaining, of self-replicating or of propagating in a host cell in order to induce the expression of a polynucleotide or of a polypeptide can be used.
• the vectors according to the invention comprise at least one origin of replication.
• the vectors of the invention also comprise at least one selection marker and preferably a selection marker usable in plant cells or in plants.
• selection markers there may be mentioned antibiotic resistance genes such as the npt11 gene for resistance to kanamycin (Bevan et al. Nature 304: 184-187, 1983) and the hph gene for resistance to 'hygromycin (Gritz et al. Gene 25: 179-188, 1983).
• herbicide tolerance genes such as the bar gene (White et al., NAR 18: 1062, 1990) for bialaphos tolerance, the EPSPS gene (US 5,188,642) for glyophosate tolerance or the HPPD gene. (WO 96/38567) for tolerance to isoxazoles. It will also be possible to use the genes coding for easily identifiable reporter enzymes such as the enzyme GUS or genes coding for pigments and enzymes regulating the production of pigments in the transformed cells. Such selection marker genes are described in particular in patent applications EP 242 236, EP 242 246, GB 2 197 653, WO 91/02071, WO 95/06128, WO 96/38567 or WO 97/04103.
• these vectors are used for the transformation of a host organism.
• Those skilled in the art will choose the appropriate transformation vectors in particular as a function of the host organism to be transformed and as a function of the transformation technique used.
• the vector for transforming plant cells or plants according to the invention is a plasmid.
• vectors have been developed for the transformation of plants with Agrobacterium tumefaciens. Other vectors are used for transformation techniques not based on the use of Agrobacterium. These vectors are well known to those skilled in the art and widely described in the literature.
• the subject of the invention is also a method of transforming host organisms, in particular plant cells with an SGS3 polynucleotide, an expression cassette or a transformation or expression vector according to the invention.
• the transformation of the host organism can be obtained by any suitable known means, the techniques of transformation and in particular of transformation of plants are amply described in the specialized literature.
• the following patents and patent applications may be cited: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604 662, EP 672,752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204.25 EP 442 174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071, WO 95/06128 and WO 99/19497.
• Agrobacterium in particular for the transformation of dicotyledons.
• a series of methods consists in using as a means of transfer into the plant a chimeric gene inserted into a Ti plasmid of Agrobacterium tumefaciens or Ri of Agrobacterium rhizogenes.
• Other methods include bombarding cells, protoplasts or tissues with particles to which the DNA sequences are attached.
• Other methods can also be used such as micro-injection or electroporation, or even direct precipitation using PEG.
• the present invention also relates to a host organism transformed with a polynucleotide SGS3, an expression cassette or a vector according to the invention.
• host organism is meant in particular according to the invention any mono or multicellular organism, lower or higher, in particular chosen from bacteria, yeasts, fungi or plant cells and plants.
• the bacteria are chosen from Escherichia coli
• the yeasts are chosen from Pichia postons and Saccharomyces cerevisae
• the fungi are chosen from Aspergillus niger.
• the host organism is a plant cell or a plant.
• Plant cell is understood according to the invention any cell derived from a plant and capable of constituting undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, plants or seeds.
• plant means any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons, more particularly crop plants intended or not for animal or human food, such as corn, wheat. barley, sorghum, rapeseed, soybeans, rice. sugar cane, beet, tobacco, cotton, clover, duckweed (lemnae) etc.
• the host organism comprises at least one other heterologous gene coding for a peptide. a polypeptide or protein of interest.
• the polynucleotide comprising a SGS3 polynucleotide according to the invention and the other heterologous gene (s) may have been introduced into the host organism simultaneously by means of the same vector comprising them, or by means of several vectors, or sequentially by means of several vectors, or by crossing several host organisms, each comprising a heterologous gene.
• heterologous gene is meant according to the invention any gene introduced artificially into the host organism, and more particularly artificially integrated into its genome, the methods allowing this introduction or integration possibly being those described above, the content of the references cited being incorporated herein by reference.
• the heterologous gene other than SGS3 polynucleotides according to the invention may be a gene comprising a coding sequence and regulatory elements at the 5 'and 3' to said coding sequence not modified with respect to the natural gene, reintroduced artificially in the genome of a host organism which may be of the same species as that from which the gene was isolated, or of a different species.
• the heterologous gene may also be a chimeric gene or an expression cassette comprising a coding sequence original, plant, bacterial, fungal, viral or animal, under the control of regulatory elements functional in the host organism, other than those naturally functionally linked to the coding sequence.
• the present invention also relates to plants containing transformed cells as defined above, in particular plants regenerated from transformed cells and leu r offspring Regeneration is obtained by any suitable process which depends on the nature of the species, as for example described in the references above.
• the present invention also relates to genetically modified plants in the genome of which an SGS3 polynucleotide or an expression cassette according to the invention are integrated in a stable and transmissible manner by sexual reproduction.
• the present invention also relates to plants obtained by crossing the regenerated plants above with other plants. It also relates to the seeds of transformed plants. Sss3 mutants
• the invention also relates to sgs3 mutants in which the SGS3 gene is inactivated. Inactivation of this gene leads to the inhibition of post-transcriptional inactivation phenomena in these mutants.
• Inactivation of the SGS3 gene in plants can be achieved by different mutagenesis techniques, site-directed mutagenesis, "machine gene” or by homologous recombination techniques (Kempin, SA et al., Targeted disruption in Arabidopsis, Nature 389: 802-803, 1997). These techniques are well known to those skilled in the art. Among the mutagenesis techniques, mention will be made of chemical mutagenesis techniques. Mention will also be made of the mutagenesis techniques using transposable elements allowing the inactivation of genes by insertion. When the mutagenesis techniques used do not specifically make it possible to inactivate the SGS3 gene, the mutants obtained are screened to identify the mutants affected in the SGS3 gene.
• This screening can be a phenotypic screening or a screening based on the amplification and sequencing of the SGS3 gene in the mutants according to techniques described in the literature.
• chimeraplasty US 6,010,907
• mutants are obtained according to the method described by Elmayan et al. (Plant Cell, 10: 1747-1757, 1998) by treating seeds with 0.4% EMS (ethyl methanesulfonate) solution. The mutants are then analyzed to identify the mutants affected in the SGS3 gene. This screening can for example be carried out by PCR.
• the present invention also relates to the use of SGS3 mutants for the identification of SGS3 genes in other plant species such as, for example, tobacco, rapeseed, sunflower, soybeans, cotton, rice, corn, sorghum, barley or wheat.
• SGS3 mutants for the identification of SGS3 genes in other plant species such as, for example, tobacco, rapeseed, sunflower, soybeans, cotton, rice, corn, sorghum, barley or wheat.
• the functional counterparts of SGS3 in other species are identified by complementing the sgs3 mutants according to the invention.
• a polynucleotide that restores the wild type of post-transcriptional inactivation is cloned.
• the sequence of this polynucleotide is then determined in order to identify the constituent elements of the cloned gene.
• the development of genetic transfer techniques has made it possible to express genes in plants, in particular with a view to improving their agronomic properties or for the production of proteins of interest.
• post-transcriptional inactivation phenomena constitute an important obstacle to the stability of the expression of transgenes in plants. These phenomena of suppression of the expression of the transgene are particularly frequent in the context of strongly expressed transgenes.
• the present invention relates to a new plant gene SGS3. Inhibition or inactivation of this SGS3 gene in plants causes inhibition of the post-transcriptional inactivation phenomenon and therefore makes it possible to obtain plants in which the expression of heterologous genes is more stable as well as plants in which the level of expression of heterologous genes is higher.
• the invention relates to a method for expressing a heterologous gene in a plant characterized in that it comprises the transformation of the plant with the heterologous gene and the inhibition of expression of the SGS3 gene in said plant.
• the invention relates to a method for expressing a heterologous gene in a plant characterized in that it comprises the following stages: a) transforming said plant with said heterologous gene; and b) inhibiting the expression of an SGS3 polynucleotide according to the invention in said plant.
• inhibiting the expression of the SGS3 gene comprises transforming the plant with a polynucleotide comprising a polynucleotide chosen from the following polynucleotides: a) an antisense polynucleotide of the coding sequence of a plant SGS3 gene; b) an antisense polynucleotide of the coding sequence of the SGS3 gene of SEQ ID NO.2; c) an expression cassette comprising, in the direction of transcription, a promoter functional in a host orgasm, a polynucleotide as defined in a) or b) and a terminator sequence functional in said host organism.
• the invention in another embodiment relates to a method for expressing a heterologous gene in a plant characterized in that it comprises the transformation of the plant with the heterologous gene and the inactivation of the expression of the SGS3 gene in said plant .
• the invention also relates to a method for expressing a heterologous gene in a plant comprising the following steps: a) transforming said plant with said heterologous gene; b) the expression of an SGS3 polynucleotide according to the invention is inactivated in said plant.
• step of inactivation or inhibition of the plant SGS3 gene and the step of transformation of the plant with a heterologous gene can be carried out simultaneously on the same plant or sequentially or by crossing several plants.
• the methods according to the invention can therefore also include stages of plant regeneration, asexual multiplication or crossing of plants.
• heterologous genes of interest can be expressed in plants in which the expression of the SGS3 gene is inhibited or inactivated.
• the heterologous gene codes for peptides, proteins or enzymes. These can be reporter proteins, selection markers or peptides or proteins of interest giving the host organism new properties, especially new agronomic properties to the transformed plants.
• genes conferring new agronomic properties on transformed plants there may be mentioned the genes conferring tolerance to certain herbicides, those conferring resistance to certain insects, those conferring tolerance to certain diseases, etc. Such genes are described in particular in patent applications WO 91/02071 and WO 95/06128.
• a gene coding for an FfPPD conferring tolerance to herbicides targeting ⁇ HPPD such as isoxazoles, in particular isoxafutole (FR 95 06800. FR 95 13570).
• diketonitriles EP 496 630, EP 496 631
• triketones in particular sulcotrione (EP 625 505, EP 625 508, US 5,506,195).
• Such genes coding for HPPD conferring tolerance to herbicides targeting HPPD are described in patent application WO 96/38567.
• proteins of interest imparting novel insect resistance properties are more particularly Bt proteins widely described in the literature and well known to the skilled person. Mention will also be made of proteins extracted from bacteria such as Photorabdus (WO 97/17432 & WO 98/08932).
• chitinases and glucanases oxalate oxidase, all these proteins and their coding sequences being widely described in the literature, or else antibacterial and / or antifungal peptides, in particular peptides of less than 100 amino acids rich in cysteines such as thionines or plant defensins, and more particularly the lytic peptides of all origins comprising one or more disulfide bridges between the cysteines and regions comprising basic amino acids, in particular the following lytic peptides: androctonine (WO 97/30082 and PCT / FR98 / 01814, deposited on August 18, 1998) or drosomicin (PCT / FR98 / 01462, filed July 8, 1998).
• the protein or peptide of interest is chosen from fungal eliciting peptides, in particular elicitins (Kamoun & al., 1993; Panab restaurants & al., 1995).
• proteins enriched in sulfur amino acids will also have the function of trapping and storing excess cysteine and / or methionine, making it possible to avoid the possible problems of toxicity linked to an overproduction of these sulfur amino acids by trapping them.
• Mention may also be made of genes coding for peptides rich in sulfur amino acids and more particularly in cysteines. said peptides also having antibacterial and / or antifungal activity.
• Mention will more particularly be made of plant defensins, as well as lytic peptides of any origin, and more particularly the following lytic peptides: androctonine (WO 97/30082 and PCT / FR98 / 01814. Deposited on August 18, 1998) or drosomicin (PCT / FR98 / 01462, filed July 8, 1998).
• the host organisms of the present invention can also be used for the production of proteins of interest in plants or "molecular farming". Indeed, the invention relates in particular to transformed plants making it possible to obtain higher levels of expression of heterologous genes.
• proteins of interest mention will in particular be made of peptides and mammalian proteins.
• immunoglobulins US 5,990,385; US 5,639,947, 5,959,177
• interferon US 4,956,282
• FIGURE 1 sgs3 Mutants
• Example 1 Isolation and Identification of the SGS3 Gene from Arabidopsis
• the mutant SGS3 (affected in the SGS3 gene) was obtained using the same experimental protocol as that which allowed the isolation of the mutants sgsl and sgs2 (Elmayan et al. Plant Cell 10: 1747-1757, 1998).
• the starting line was the Ll line.
• Ll is a transgenic line obtained by transformation of plants of the Columbia ecotype by construction 23b (Elmayan and Vaucheret, Plant J. 9: 787-797, 1996).
• the L1 line has only one transgenic locus.
• the glucuronidase activity in the L1 line is 4000 nmol of 4-methylumbelliferone per minute and per microgram of total proteins in the first days of development.
• the GUS activity in these 6 mutant lines, 1 month after germination, is between 2500 and 3500 nmol of 4-methylumbelliferone per minute and per microgram of total proteins. .
• the GUS activity in these mutant lines, 1 month after germination, is between 2,500 and 3,500 nmol of 4-methylumbelliferone per minute and per microgram of total proteins.
• the GUS activity, the mRNA accumulation and the transcription rate were measured by fluorimetric tests, by blot analysis. mRNA and through "run-on" experiences.
• the GUS activity is multiplied by a factor of 300 in the sgs2 mutants compared to the L1 line while the accumulation of mRNA is multiplied by a factor of 250.
• the transcription rate is only multiplied by a factor 2.6 compared to the Ll line.
• Line 2a3 is a transgenic line of Arabidopsis thaliana resulting from the transformation of a plant of the Columbia ecotype by construct 2a (Elmayan et al., Plant Cell 10: 1747-1757, 1998) containing the transcribed part of the gene NIA2 from Arabidopsis encoding nitrate reductase under the control of the 35S promoter and the hygromycin hpt resistance gene. All the plants of the 2a3 line homozygous for the 2a construction have a post-transcriptional inactivation of the Nia2 genes (transgenic and endogenous) leading to chlorosis of the plant and then to its death.
• the transgenic locus 2a3 When the transgenic locus 2a3 is in the heterozygous state, only part of the plants undergo post-transcriptional inactivation. The stage at which this inactivation takes place varies from one plant to another. In some plants inactivation is late enough to allow the production of pollen and seeds.
• the hybrid plants resulting from the cross between the mutant sgs3-2 and the line 2a3 were cultivated in the greenhouse and the seeds for self-fertilization were harvested. These were sown in the greenhouse and the plants obtained without chlorosis were kept to harvest their seeds for self-fertilization. We then sowed the different lots of seeds on an agar medium containing 20 mg / l of hygromycin.
• sgs3-1 mutants were inoculated with the cucumber mosaic virus (CMV) strain II 7F.
• CMV cucumber mosaic virus
• infection with this viral strain leads to plants whose development is slower and altered: smaller rosette leaves, long but very flexible flower stalk, reduced but not zero fertility.
• infection with this viral strain leads to an increased alteration of development: the plants have a particularly bushy habit, the leaves of the rosette are small and twisted, the flower stalk reaches a size at the end of development of the order of 5 cm, the plants are completely sterile.
• F2-1 lines 120 homozygous F2 lines for the sgs3- ⁇ mutation
• F2-2 lines 90 homozygous F2 lines for the sgs3-2 mutation
• the polymorphism corresponding to the molecular marker 13H2L was revealed by hybridization (of the Southern blot type) of the total DNA of Arabidopsis plants, digested with the restriction enzyme HindIII, by a fragment of radioactive DNA corresponding to l left end of the artificial yeast chromosome (YAC) 13H2 (probe 13H2L).
• the polymorphism corresponding to the molecular marker 3B3D was revealed by hybridization (of the type
• the resulting plasmids were introduced into E. coli and then into the Agrobacterium tumefaciens C58pMP90 strain.
• the resulting bacterial strains were used to transform plants of the sgs3-2 2a3 lines.
• Strain Bacterial 356 made it possible to obtain 20 transgenic lines. Among these 20 lines. 19 showed signs of chlorosis identical to those observed on line 2a3. On 3 of these plants we were able to show by northem type hybridization using as probe NIA2 gene from Arabidopsis thaliana.
• the DNA sequence inserted at the BamHI site of the plasmid pBin + and which led to the isolation of the bacterial strain 356 was determined. Subclones of clone 356 were made in the vector pBin + and the same sgs3-2 2a3 line was transformed by these subclones in order to determine those capable of restoring the function of the SGS3 gene. The smallest subclone capable of restoring this function constitutes the SGS3 gene as described in this patent. By computer analysis the ORS of SGS3 could be predicted. The sequence of the cDNA containing the ORF of the SGS3 gene and therefore the position of the promoter, terminator and intronic sequences of SGS3 were verified after having isolated and cloned this sequence.
• PCR was carried out on the genomic DNA of these 5 mutants using the primers p356AD 'and P356Y' (see example 1). This reaction allows the amplification of the entire SGS3 gene. The fragment amplified by this PCR reaction was sequenced.
• the nucleotide sequence thus obtained is then treated with the enzyme “klenow” to generate “blunt” ends at the ends of the amplified sequence.
• the sequence is then cloned between the 35S promoter and the cauliflower mosaic virus terminator at the Smal site of the vector pRTl 00.
• the clones are selected such that the sequence corresponding to p356AD 'is located near the 35S promoter.
• clones are selected such that the sequence corresponding to p356Y 'is located near the 35S promoter.
• This Assgs3 construction allows the expression of an anti-sense mRNA of the SGS3 mRNA.
• Example 4 Transformation of Plants The expression cassettes constructed as described above are then introduced into a binary vector to allow their introduction via Agrobacterium tumefaciens into plants.
• the binary vector used is the plasmid pBIN + (Van Engelen et al., Transgenic Research 4, 288-290, 1995). This is done by digesting the constructions obtained above with the enzyme SphI (which releases the expression cassettes) and by ligating the product of this digestion with the plasmid pBIN + digested with the enzyme SphI.
• Example 5 Inhibition of Expression of the SGS3 Gene by Antisense
• the complete cDNA of the SGS3 gene was cloned in antisense orientation (aSGS3) between the 35S promoter (p35S) and the 35S terminator (t35S).
• the chimeric gene p35S-aSGS3-t35S was re-cloned into the binary vector pBiB-Hyg then transferred into Agrobacterium tumefaciens.
• Plants of the L1 line p35S-GUS-tRbcS gene subjected to PTGS
• the transformed plants were selected on medium supplemented with hygromycin.
• the GUS activity of the p35S-GUS-tRbcS transgene was measured in the untransformed L1 plants, in 28 hygromycin-resistant transformants, as well as in the sgs3 mutants obtained by EMS mutagenesis of the L1 line.
• the GUS activity in untransformed L1 plants is between 0 and 10 nmol MU / min / ug of protein while the GUS activity in sgs3 mutants is between 3000 and 5500 nmol MU / min / ug of proteins.

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