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)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• • 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
• • 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)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition

Definitions

• the present invention relates to the field of improving the agronomic characteristics of plants, with a view to obtaining plant transformation products with improved characteristics for industry, especially for the food industry, for example for the production of starch, vegetable oil or semolina.
• the seed embryo is the seed compartment from which vegetable oil is extracted. It would be advantageous for the industry to obtain plants whose seeds have an overdeveloped embryo rich in oil.
• Starch production for multiple industrial applications today reaches around 1 billion tonnes annually worldwide.
• Starch represents a raw material which is used in industries as diverse as food, pharmacy, paper industries but also in the microbiological field where it can be used as a nutritive substrate.
• starch is obtained from seeds, from cereal crops grown in fields such as wheat, corn and sorghum.
• the seeds are essentially made up of a germ associated with albumen.
• the starch is entirely contained in the endosperm, while the germ is rich in oil.
• the starch preparation processes used in the starch industry necessarily include a step of separation of the germ, rich in oil, from the albumen which contains in abundance the starch constituting the
• the milling process allows the different constituents of the grain to be finely separated in order to meet user requirements. It includes in particular a drying step which must be as gentle as possible to avoid a migration of lipids from the germ to the almond, which is detrimental to the subsequent quality of the semolina, and a degerming step, carried out by fragmentation, which has intended to gently separate the germs to prevent the lipids they contain from being found in the semolina.
• the use in semolina industry of grains with development of germ affected according to the invention therefore has a certain advantage since it facilitates the industrial process (gain in time and in yield) by eliminating this 'contamination' of the kernel by the germ lipids; moreover, it ensures good quality and good shelf life of the products, this being a function of the residual fat content ( ⁇ 0.8% is a standard in the profession for so-called noble products: hominies, gritz, semolina and flours for human food).
• the semolina produced is then used almost exclusively in human food (beers, breakfast cereals, aperitif cookies, polenta, tortilla, corn chips, edible flour ).
• the grains according to the invention can be used for the manufacture of breakfast cereals or 'corn flakes 1 , which constitute a market which has known for 15 years an annual average increase of the order of 20%.
• Two processes are conventionally used: a conventional rolling process from hominys (the largest semolina perfectly degermed and calibrated) or a cooking-extrusion process from semolina of specific particle size.
• hominys the largest semolina perfectly degermed and calibrated
• a cooking-extrusion process from semolina of specific particle size.
• seeds with large embryos, which are sought after in the oil industry can be obtained.
• Corn oil is generally intended for human consumption; it is sometimes used by the pharmaceutical and cosmetic industry.
• the cakes are themselves valued in animal feed either directly, or even remixed with 'corn-gluten feed'.
• mutant plants carrying mutations affecting both the embryo and the seed albumen are known in the state of the art. These mutant plants are conventionally designated as “dek” mutants (for “detective kernel”). On the other hand, there are very few corn mutants affected by a defect in the development of the seed embryo and leaving the albumen intact.
• a first group of mutants includes mutants producing proembryonic structures resembling those produced in wild plants, but the proembryos do not reach later stages of development.
• the mutants emb * -8522 and emb * -8535 are affected by a complete deficit in apical-basal differentiation, while in the mutant emb * -8516 the authors observed structures resembling l embryo that came from the suspensor.
• a co-segregation analysis of the emb * -8516 and emb * -8522 mutations enabled Heckel et al. to determine that the mutant phenotype co-segregated with the presence of a transposon.
• the molecular characterization of the different mutations has not been described.
• the mutation affecting the mutant plant emb * -8516 could not be located on any of the corn chromosomes.
• nucleic acid sequences and polypeptides are provided, the expression of which is essential for the development of the embryo in a plant seed, and of which a deficit in expression leads to the production of affected seeds. in the development of the germ.
• the subject of the invention is a nucleic acid comprising a polynucleotide coding for an ISU 2A polypeptide chosen from sequences having at least 95% amino acid identity with the sequences SEQ ID No. 5 and SEQ ID No. 6, or for a fragment of an ISUM2A polypeptide, as well as a nucleic acid of complementary sequence.
• the nucleic acid coding for the ISUM2A polypeptide, or for a fragment of this polypeptide also comprises a regulatory polynucleotide capable of regulating the synthesis of the ISUM2A polypeptide, the regulatory polynucleotide preferably being sensitive to the action of an inducing signal.
• the regulatory polynucleotide can be either a transcription or translation repressing polynucleotide or, on the contrary, a transcription or translation activating polynucleotide.
• the invention also relates to methods for obtaining a transformed plant capable of producing seeds with development of an affected germ, as well as the parts of such a plant, in particular its seeds.
• the invention also relates to a transformation product of a seed with development of the affected germ produced by said transformed plant, preferably a starch.
• a transformation product of a seed with development of the affected germ produced by said transformed plant preferably a starch.
• Another subject of the invention is the ISUM2A polypeptide, or a fragment of this polypeptide, encoded by a nucleic acid as defined above, as well as antibodies directed against the ISUM2A polypeptide.
• the invention also relates to methods for detecting the presence of the ISU 2A polypeptide in a sample and to methods for detecting the presence of a nucleic acid encoding this polypeptide in a sample.
• a population of 25,000 highly mutagenized plants has been generated by the random insertion, into their genome, of the transposon m ⁇ tator described by Bennetzen, JLPS Springer, AD Cresse, and M. Hendrickx (1993), Chandler, VL and KJ Hardeman (1992).
• the plants were cultured before analysis of their DNA.
• G2422 it has been shown that the insertion of the mutator transposon was localized in the first intron of a particular gene, this gene having been designated isum2A, at a distance of 3 bp from the second exon of this gene.
• the inventors observed a co-segregation between the presence of a “seed without germ” phenotype and the insertion of the mutator transposon in the same isum2A gene.
• the is ⁇ m2A gene includes three exons and two introns.
• the open reading frame begins in the first exon and ends in the third exon of this gene.
• a partial transcript of the isum2A gene was also isolated and characterized, and the structure of a large portion of the ISUM2A protein was deduced from the cDNA corresponding to the gene transcript.
• isum2A gene is expressed in the embryo and the endosperm 12 days after pollination, as well as in the leaf and the root.
• ISUM2A produced seeds essentially consisting of albumen, and having an affected germ development.
• the germ is also called an embryo.
• the applicant has also shown that the seeds which represent a quarter of the seeds produced by heterozygous plants and of which the two copies of the isum2A gene include a mutation do not allow the obtaining of a viable and fertile plant.
• the invention also provides the means for obtaining plants producing seeds affected in the development of the germ, in which the germ is enriched in oil due to early expression or overexpression of the polypeptide.
• the invention relates to nucleotide sequences involved in the development of the embryo and their use in molecular constructions intended to improve the agronomic, food, or industrial quality of a plant, in particular modulating the size of the embryo and / or its size. development.
• an early and specific action on the development of the tissues of the embryo and the endosperm can be sought: 1) According to a first embodiment, it will be possible to obtain seeds or fruits enriched in oil (large embryo), via the use of promoters directing the expression of the nucleic acids according to the invention early in the development of the seed and more particularly in the germ, or in a constitutive manner. 2) According to another embodiment, albumens with development of affected germ could also be obtained according to this model, for industrial applications in starch manufacture and semolina. The invention therefore also relates to methods for modifying the agronomic and / or nutritional qualities of a plant, by a targeted and early action on the development of the embryo, using the transformation of plants with a vector according to the invention.
• she is interested in modifying the size and / or development of the embryo. It also aims at altering the development of the embryo, with a view to producing grains without embryos for cereals in particular, which are of interest for the starch and semolina industries.
• the subject of the invention is also the use of an expression cassette as defined above, for obtaining a transgenic Angiosperm plant having improved agronomic or nutritional qualities.
• the transgenic plant obtained can produce seeds with modified oil contents or with development of an affected germ in comparison with an unprocessed plant.
• the invention also relates to the use of the transgenic plants obtained according to the invention, or parts of these plants, in particular seeds, grains and fruits for the preparation of derived products, in particular food products.
• Also part of the invention are the products obtained, whether seeds, seed flour or grains enriched in oil or seeds with development of affected germ, suitable for the semolina industry.
• the invention also relates to any composition for human or animal food prepared from said products obtained.
• the invention relates to the use of the allelic sequences and variants defined according to the invention in selection programs aimed at producing plants with an embryo modified in size and / or development influencing the starch content / oil.
• selection programs aimed at producing plants with an embryo modified in size and / or development influencing the starch content / oil.
• These sequences can in particular be used in QTL mapping and co-localization experiments for the oil content or the size of the embryo to define the most interesting allelic variants for a selection approach, comprising:
• nucleotide sequence of the isum2A gene comprises, from the 5 ′ end to the 3 ′ end, (i) an upstream non-coding sequence potentially carrying regulatory elements for the transcription and / or translation of the gene, (ii) a coding sequence comprising the three exons and the two introns of the gene and (iii) a non-coding sequence located downstream of the last exon of the gene.
• sequence of the isum2A gene according to the invention is referenced as the sequence SEQ ID No. 1 of the sequence listing.
• An analysis of a population of plants having the wild phenotype and producing seeds containing a normal embryo made it possible to identify at least two variants of the isum2A gene.
• One of the gene variants is characterized by the substitution of the nucleotide G located at position 2234 of the sequence SEQ ID No. 1 by a nucleotide C. This substitution of nucleotide leads to the substitution of the amino acid G (glycine) at position 89 of the sequence of the ISUM2A SEQ ID No. 5 polypeptide by an amino acid R (Asparagine) which is present in the sequence of the ISUM2A polypeptide variant SEQ ID No. 6.
• a first object of the invention consists of a nucleic acid comprising a polynucleotide encoding a polypeptide
• ISUM2A chosen from sequences having at least 95% amino acid identity with the sequences SEQ ID No. 5 and SEQ ID No. 6, or for a fragment of an ISUM2A polypeptide.
• the invention also relates to a nucleic acid of sequence complementary to the nucleic acid as defined above.
• any conventional technique of molecular biology, microbiology and recombinant DNA known to those skilled in the art can be used. Such techniques are described for example by SAMBROOK et al. (1989), GLOVER (1985), GAIT (1984),
• any nucleic acid and any polypeptide according to the invention is in an isolated or purified form.
• isolated in the sense of the present invention designates a biological material which has been removed from its original environment (the environment in which it is naturally located).
• a polynucleotide found naturally in a plant is not isolated.
• the same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted into the genome of the plant is isolated.
• Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment.
• purified does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
• a polynucleotide or a polypeptide is in the purified state after purification of the starting material or of the natural material of at least one order of magnitude, preferably 2 or 3 and preferably four or five orders of magnitude.
• nucleotide sequence can be used to denote either a polynucleotide or a nucleic acid.
• nucleotide sequence encompasses the genetic material itself and is therefore not limited to information regarding its sequence.
• nucleic acid include RNA, DNA, cDNA sequences or even RNA / DNA hybrid sequences of more than one nucleotide, in single strand form or in duplex form.
• nucleotide designates both natural nucleotides (A, T, G, C) as well as modified nucleotides which comprise at least one modification such as (i) an analog of a purine, (ii) an analog of d 'a pyrimidine, or (iii) a similar sugar, such modified nucleotides being described for example in PCT application No. WO 95/04064.
• a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired with the complementary base of the second polynucleotide whose orientation is reversed.
• the complementary bases are A and T (or A and U), and C and G.
• a first nucleic acid having at least 95% identity with a second reference nucleic acid will have at least 95%, preferably at least 96%, 97%, 98%, 98.5%, 99 %, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity nucleotides with this second reference polynucleotide, the percentage of identity between two sequences being determined as described below.
• the "percentage of identity" between two nucleotide or amino acid sequences can be determined by comparing two optimally aligned sequences, through a comparison window.
• the part of the nucleotide or polypeptide sequence in the comparison window can thus include additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment of the two sequences.
• the percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues compared, by the total number of positions in the comparison window, then multiplying the result by one hundred to obtain the percentage of sequence identity.
• the optimal alignment of the sequences for the comparison can be carried out by computer using known algorithms.
• the percentage of sequence identity is determined using the BLAST software (BLAST version 2.06 of September 1998), using exclusively the default parameters.
• a nucleic acid having at least 95% nucleotide identity with a nucleic acid according to the invention includes the "variants" of a nucleic acid according to the invention.
• variant of a nucleic acid according to the invention is meant a nucleic acid which differs from the reference nucleic acid by one or more substitutions, additions or deletions of a nucleotide, relative to the nucleic acid of reference.
• a variant of a nucleic acid according to the invention can be of natural origin, such as an allelic variant which exists naturally. Such a variant nucleic acid can also be an unnatural nucleic acid obtained, for example, by mutagenesis techniques.
• the differences between the reference nucleic acid and the "variant" nucleic acid are reduced so that the reference nucleic acid and the variant nucleic acid have very similar nucleotide sequences and, in many regions , identical.
• the nucleotide modifications present in a variant nucleic acid can be silent, which means that they do not affect the amino acid sequence which can be encoded by this variant nucleic acid.
• Changes in nucleotides in the variant nucleic acid can also result in substitutions, additions or deletions of one or more amino acids in the sequence of the polypeptide which can be encoded by this variant nucleic acid.
• a variant nucleic acid according to the invention comprising an open reading phase, code for a polypeptide which retains the same function or the same biological activity as the polypeptide coded by the reference nucleic acid.
• a variant nucleic acid according to the invention and which comprises an open reading phase codes for a polypeptide which retains the capacity to be recognized by antibodies directed against the polypeptide encoded by the nucleic acid of reference.
• nucleic acids of genes orthologous to ISUM2 included in the genome of plants other than maize, and having a nucleotide identity of at least 95% with a nucleic acid are part of the “variants” of a nucleic acid encoding the ISUM2A polypeptide. encoding the ISUM2A polypeptide.
• fragment of a nucleic acid according to the invention is meant a nucleotide sequence of a reduced length compared to the reference nucleic acid, the nucleic acid fragment having a nucleotide sequence identical to the nucleotide sequence of the reference nucleic acid on the common part.
• fragments of a nucleic acid according to the invention have at least 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300 , 400, 500, 1000, 2000 or 3000 consecutive nucleotides of the reference nucleic acid, the maximum length in nucleotides of a fragment of a nucleic acid according to the invention is of course limited by the maximum length in nucleotides of l reference nucleic acid.
• fragment of an ISU 2A polypeptide according to the invention means a polypeptide fragment of reduced length compared to the reference polypeptide, the polypeptide fragment having an amino acid sequence identical to the amino acid sequence of the polypeptide of reference on the common part.
• fragments of an ISUM2A polypeptide according to the invention have at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 135 or 140 amino acids of a reference ISUM2A polypeptide.
• allelic variants of the genomic nucleic acid of the isum2A gene have been characterized according to the invention, the two variant genomic nucleic acids being respectively referenced as the nucleotide sequences SEQ ID No. 1 and SEQ ID No. 2 of the sequence listing.
• the subject of the present invention is a nucleic acid coding for an ISUM2A polypeptide, or for a fragment of this polypeptide, said nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a nucleotide sequence chosen from SEQ ID N ° 1 and SEQ ID N ° 2, or with a fragment of one of the sequences SEQ ID N ° 1 and SEQ ID N ° 2.
• nucleic acid of sequence complementary to the nucleic acid as defined above.
• nucleic acid consisting of a polynucleotide having at least 95% nucleotide identity with a sequence chosen from the sequences SEQ ID No 1 and SEQ ID No 2, or with a fragment of one of the sequences SEQ ID No 1 or SEQ ID No 2, or a nucleic acid of complementary sequence.
• the invention also relates to a nucleic acid comprising at least 12, preferably at least 15 and very preferably at least 20 consecutive nucleotides of the nucleic acid of sequence SEQ ID No. 1 or SEQ ID No. 2 , it being understood that such a nucleic acid includes in its definition the "fragments" of a nucleic acid according to the invention as defined in the present description.
• the isum2A gene defined by the sequence SEQ ID No. 1 comprises, from the 5 ′ end to the 3 ′ end, respectively: a) a non-coding sequence potentially carrying regulatory elements for transcription and / or translation of this gene, located upstream of the first exon, from the nucleotide in position 1 to the nucleotide in position 1812 of the sequence SEQ ID No.
• coding region which comprises the three exons and the two introns of the isum2A gene, this coding region being located from the nucleotide at position 1813 to the nucleotide at position 4074 of the sequence SEQ ID No. 1; and c) a non-coding region located downstream of the coding region, from the nucleotide at position 4075 to the nucleotide at position 5620 of the sequence SEQ ID No. 1.
• sequence SEQ ID No. 2 which illustrates a variant of the isum2A gene, does not contain the entire open reading frame coding for an ISU 2A polypeptide.
• the sequence SEQ ID N ° 2 includes a part located on the 3 ′ side of exon n ° 1, as well as all of exons n ° 2 and n ° 3.
• the nucleotide at position 1 of the sequence SEQ ID No. 2 corresponds to the nucleotide at position 2063 of the sequence SEQ ID No. 1.
• the nucleotide at position 2004 of the sequence SEQ ID No. 2 corresponds to the nucleotide at position 4062 of the sequence SEQ ID No. 1.
• sequence SEQ iD No. 2 by those skilled in the art, and its comparison with the sequence SEQ ID No. 1, allows direct access to the entire coding sequence of the isum2A gene defined by the sequence SEQ ID N ° 2, for example by completing the missing bases of exon n ° 1 of the sequence SEQ ID N ° 2 by the corresponding bases of exon n ° 1 of the sequence SEQ ID N ° 1, based on the correspondences given above and also in table 1 below.
• nucleic acid of the isum2A gene for example by isolating, from the information of the sequence SEQ ID No. 1, the equivalent nucleotide regions or else by producing a hybrid nucleic acid, obtained by fusing the nucleic acids of sequences SEQ ID No. 1 to SEQ ID No. 2, on the basis of the correspondences given above and also in table 1 below.
• Such a nucleic acid codes for at least part of the polypeptide encoded by the lsum2A gene and can in particular be inserted into a recombinant vector intended for the expression of the corresponding translation product in a host cell or in a plant transformed with this recombinant vector.
• Such a nucleic acid can also be used for the synthesis of nucleotide probes and primers intended for the detection or the amplification of nucleotide sequences included in the lsum2A gene in a sample, if necessary of gene sequences.
• lsum2A carrying one or more mutations, preferably one or more mutations likely to modify the phenotype of a plant carrying such a mutated lsum2A gene, by causing the production of affected seeds in the development of the germ.
• the invention also relates to a nucleic acid comprising at least 12 consecutive nucleotides of an intronic polynucleotide of the lsum2A gene, such as the polynucleotides 1 and 2 described in table 2 above, which are included in the nucleic acid of sequence SEQ ID N ° 1 and SEQ ID N ° 2.
• a nucleic acid can be used as an oligonucleotide probe or primer to detect the presence of at least one copy of the lsum2A gene in a sample, or even to amplify a determined target sequence within the lsum2A gene.
• Such a nucleic acid can also be used to amplify a specific target sequence within the isum2A gene or to inhibit it by a sense or co-suppression approach, or by the use of double-stranded RNA (Wassenegger et al. 1996; Kooter et al. 1999) for interference.
• Such a nucleic acid can also be used to search for functional allelic variants of the ISUM2 gene, which can be used in a method for selecting plants with an embryo modified in size and / or development.
• the genomic region of the lsum2A gene essentially restricted to the so-called "coding" region comprising the 3 exons and the 2 introns is defined as the sequence starting at the nucleotide at position 1813 and ending at the nucleotide at position 4074 of the sequence SEQ ID No. 1 .
• Part of exon n ° 1, all exons n ° 2 and 3 as well as the two introns of a .variant of the isum2A gene are included in the sequence going from the nucleotide in position 1 to the nucleotide in position 2004 of the sequence SEQ ID N ° 2. It is specified that, on their common region, the sequences
• SEQ ID N ° 1 and SEQ ID N ° 2 have a percentage of nucleotide identity greater than 95%, this percentage being in fact greater than 99%.
• the subject of the invention is also a nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with the nucleotide sequence starting at the nucleotide in position 1813 and ending at the nucleotide in position 4074 of the sequence SEQ ID N ° 1 as well than a nucleic acid of complementary sequence.
• the invention also relates to a nucleic acid having at least 95% nucleotide identity with the nucleotide sequence starting at the nucleotide at position 1813 and ending at the nucleotide at position 4074 of the sequence SEQ ID No. 1, as well as an acid nucleic acid of complementary sequence.
• the subject of the invention is also a nucleic acid comprising the nucleotide sequence starting at the nucleotide at position 1813 and ending at the nucleotide at position 4074 of the sequence SEQ ID No. 1 or a nucleic acid with complementary sequence.
• the invention also relates to a nucleic acid consisting of the nucleotide sequence starting at the nucleotide at position 1813 and ending at the nucleotide at position 4074 of the sequence SEQ ID No. 1 or a nucleic acid of complementary sequence.
• Another object of the invention consists of a nucleic acid characterized in that it comprises one of the following nucleotide sequences: a) the sequence going from the nucleotide in position 1 to the nucleotide in position 1812 of the sequence SEQ ID N ° 1, or a nucleic acid of complementary sequence; b) the sequence extending from the nucleotide at position 1813 to the nucleotide at position 2099 of the sequence SEQ ID No. 1, or a nucleic acid of complementary sequence; c) the sequence going from the nucleotide at position 2100 to the nucleotide at position 2206 of the sequence SEQ ID No.
• nucleic acid of complementary sequence 1, or a nucleic acid of complementary sequence; d) the sequence going from the nucleotide in position 2207 to the nucleotide in position 2323 of the sequence SEQ ID No. 1, or a nucleic acid of complementary sequence; e) the sequence going from the nucleotide at position 2324 to the nucleotide at position 3645 of the sequence SEQ ID No. 1, or a nucleic acid of complementary sequence; f) the sequence going from the nucleotide at position 3646 to the nucleotide at position 4074 of the sequence SEQ ID No. 1, or a nucleic acid of complementary sequence; and g) the sequence going from the nucleotide at position 4075 to the nucleotide at position 5620 of the sequence SEQ ID No. 1 or a nucleic acid of complementary sequence.
• nucleic acid of sequence SEQ ID No. 1 is shown in FIG. 1, in which the positions of the various exons and introns of the lsum2A gene are also detailed.
• the lsum2A gene is transcribed in the form of a messenger RNA.
• This messenger RNA includes an open reading frame coding for the protein ISUM2A
• the part of the cDNA of the lsum2A gene comprising the open reading frame coding for the polypeptide ISUM2A of sequence SEQ ID No. 5 has a length of 833 nucleotides and is referenced like the sequence SEQ ID N ° 3 of the sequence listing.
• the cDNA of sequence SEQ ID No. 3 is derived from the wild plant variant designated HD5xHD7. This cDNA is shown in Figure 2.
• the part of the cDNA of the lsum2A gene comprising a part of the open reading frame coding for the ISUM2A polypeptide of sequence SEQ ID No. 6 has a length of 621 nucleotides and is referenced as the sequence SEQ ID No. 4 of the sequence listing .
• Sequence cDNA SEQ ID No. 4 is derived from the wild plant variant designated A188. This partial cDNA is shown in Figure 3.
• nucleic acids of sequences SEQ ID No. 3 and SEQ ID No. 4 show similarities to an EST sequence referenced in the GENBANK database under the access number AI001298 and designated “ISUM2”. This is the reason why the designation “isum2A” has been attributed to the newly discovered gene according to the invention.
• sequence of EST N ° AI001298 has a degree of nucleotide identity of 94% and 95% with the sequences SEQ ID N ° 3 and SEQ ID N ° 4, respectively. No open reading frame is described for this EST.
• sequences SEQ ID No. 3 and SEQ ID No. 4 also exhibit similarities with an EST sequence referenced in the GENBANK database under the access number AI374506. This sequence was obtained from the same clone “EST6-D3” as the sequence AI 001298.
• sequence of EST n ° AI374506 has a degree of nucleotide identity of 92% and 98% with the sequences SEQ ID N ° 3 and SEQ ID N ° 4, respectively. No open reading frame is described for this EST.
• Another subject of the invention consists of a nucleic acid comprising a polynucleotide coding for an ISUM2A polypeptide and having at least 99% nucleotide identity with the nucleotide sequence SEQ ID No. 3 or SEQ ID No. 4, or with a fragment of this nucleotide sequence as well as a nucleic acid of sequence complementary to these nucleic acids.
• the invention also relates to a nucleic acid coding for an ISUM2A polypeptide and having at least 99% nucleotide identity with the nucleotide sequence SEQ ID No. 3 or SEQ ID No. 4 or a fragment of this nucleotide sequence as well as 'a nucleic acid of sequence complementary to these nucleic acids.
• the subject of the invention is also a nucleic acid characterized in that it comprises the nucleotide sequence SEQ ID No. 3 or SEQ ID No. 4 or a nucleic acid of complementary sequence. Also part of the invention is a nucleic acid consisting of the nucleotide sequence SEQ ID No. 3 or SEQ ID No. 4, or a nucleic acid of complementary sequence.
• the lsum2A gene codes for a polypeptide of 143 amino acids in length, of which two allelic variants have been identified according to the invention, the variant polypeptides of sequence SEQ respectively.
• the invention also relates to a nucleic acid coding for a polypeptide having at least 95% identity in amino acids with the sequence SEQ ID No. 5 or SEQ ID No. 6.
• the invention also relates to a nucleic acid characterized in that it codes for the polypeptide of sequence SEQ ID No. 5 or SEQ ID No. 6. Also part of the invention is a nucleic acid encoding a "fragment" of a polypeptide having at least 95% nucleotide identity with a polypeptide of amino acid sequence SEQ ID No. 5 or SEQ ID No. 6.
• the invention also relates to a nucleic acid coding for a fragment of a polypeptide with an amino acid sequence SEQ ID No. 5 or SEQ ID No. 6.
• a polypeptide having at least 95% amino acid identity with a reference polypeptide comprises at least 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% of amino acid identity with the reference polypeptide.
• variant polypeptides are included in the definition of a polypeptide having at least 95% amino acid identity with a reference polypeptide according to the invention, the so-called "variant” polypeptides.
• the term “variant” of a polypeptide according to the invention means a polypeptide whose amino acid sequence comprises one or more substitutions, additions or deletions of at least one amino acid residue, relative to the sequence of amino acids of the reference polypeptide, it being understood that the amino acid substitutions can be of a conservative or non-conservative nature.
• a variant of a reference polypeptide according to the invention consists of a polypeptide which retains the biological function or activity of the reference polypeptide and / or which is recognized by antibodies directed against the reference polypeptide. These polypeptide variants can result from allelic variations characterized by differences in the nucleotide sequences of the gene coding for these polypeptides. Such polypeptide variants can also result from alternative splicing or from post-translational modifications.
• fragment of a reference polypeptide according to the invention is meant a polypeptide having at least 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 135 or 140 consecutive amino acids of a polypeptide as defined in the present description.
• nucleic acids according to the invention and in particular the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 4, their fragments of at least 12 nucleotides, the sequences having at least 95% identity in nucleotides with at least part of the sequences SEQ ID N ° 1 to SEQ ID N ° 4, as well as the nucleic acids of complementary sequence, are useful for detecting the presence of at least one copy of a nucleotide sequence of the lsum2A gene or else d 'a fragment or an allelic variant of the latter in a sample.
• nucleotide probes and primers which hybridize, under high stringency hybridization conditions, with a nucleic acid chosen from the sequences SEQ ID No. 1 to SEQ ID No. 4.
• hybridization conditions below are implemented for the hybridization of a nucleic acid, probe or primer, 20 bases in length.
• the level and specificity of hybridization depends on various parameters, such as: a) the purity of the preparation of the nucleic acid on which the probe or the primer is to hybridize; b) the base composition of the probe or the primer, the GC base pairs having greater thermal stability than the A-T or AU base pairs; c) the length of the sequence of homologous bases between the probe or the primer and the nucleic acid; d) ionic strength: the rate of hybridization increases with increasing ionic strength and the duration of the incubation time; e) the incubation temperature; f) the concentration of the nucleic acid on which the probe or the primer is to hybridize; g) the presence of denaturing agents such as agents promoting the breaking of hydrogen bonds, such as formamide or urea, which increase the stringency of the hybridization; h) the incubation time, the rate of hybridization increasing with the duration of the incubation; i) the presence of bulk exclusion agents, such as dextran or dextran sulfate, which
• the parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
• Tm is defined by the relation:
• Tm 81.5 + 0.41 (% G + C) +16.6 Log (cation concentration) - 0.63 (% formamide) - (600 / number of bases) (SAMBROOK et al., (1989) , pages 9.54-9.62).
• the hybridization temperature is approximately 5 to 30 ° C, preferably 5 to 10 ° C below Tm.
• high stringency hybridization conditions hybridization conditions such that one places oneself at a hybridization temperature of 5 ° C. below the Tm.
• hybridization conditions described above can be adapted as a function of the length and of the base composition of the nucleic acid whose hybridization is sought or of the type of labeling chosen, according to techniques known to those skilled in the art. job.
• hybridization conditions used for a nucleic acid 200 bases in length are as follows: Prehybridization: same conditions as for hybridization duration: 1 night.
• the nucleotide probes or primers according to the invention comprise at least 12 consecutive nucleotides of a nucleic acid according to the invention, in particular of a nucleic acid of sequences SEQ ID N ° 1 to 4 or of its complementary sequence, a nucleic acid having 95% nucleotide identity with a sequence chosen from sequences SEQ ID No. 1 to 4 or. of its complementary sequence or also of a nucleic acid hybridizing under hybridization conditions of high stringency with a sequence chosen from sequences SEQ ID No. 1 to 4 or of its complementary sequence.
• nucleotide probes or primers according to the invention will have a length of at least 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 100, 150, 200, 300, 400, 500 , 1000, 2000 or 3000 consecutive nucleotides of a nucleic acid according to the invention.
• a probe or a nucleotide primer according to the invention will consist and / or include fragments with a length of 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 100, 150, 200, 300, 400, 500, 1000, 2000 or 3000 consecutive nucleotides of a nucleic acid according to the invention.
• primers and of pairs of primers making it possible to amplify a nucleic acid fragment of the lsum2A gene of sequence SEQ ID No 1 or SEQ ID No 2 are for example primers SEQ ID No 10 to 13 and 17 to 23.
• a primer or a nucleotide probe according to the invention can be prepared by any suitable method well known to those skilled in the art, including by cloning and action of restriction enzymes or also by direct chemical synthesis according to techniques such as the method to the phosphodiester of NARANG et al. (1979) or BROWN et al. (1979), the diethylphosphoramidite method of BEAUCAGE et al. (1980) or the solid support technique described in European patent No. EP 0 707 592.
• Each of the nucleic acids according to the invention can be labeled, if desired, by incorporating a detectable molecule, that is to say a detectable marker, by spectroscopic, photochemical, biochemical, immunochemical or even chemical means.
• markers can consist of radioactive isotopes ( 32 P 3 H, 35 S), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetylaminofluorene) or also ligands such as biotin.
• the labeling of the probes is preferably done by incorporating labeled molecules within the polynucleotides by extension of primers, or else by adding to the 5 ′ or 3 ′ ends.
• oligonucleotide probes according to the invention can be used in particular in Southern type hybridizations to the genomic DNA of the lsum2A gene or else in hybridizations to messenger RNA of this gene when the expression of the corresponding transcript is sought in a sample.
• the probes according to the invention can also be used for the detection of PCR amplification products or even for the detection of mismatches.
• Nucleotide probes or primers according to the invention can be immobilized on a solid support.
• Such solid supports are well known to those skilled in the art and comprise surfaces of the wells of microtitration plates, polystyrene beads, magnetic beads, nitrocellulose strips or even microparticles such as latex particles.
• the subject of the invention is also a nucleic acid which can be used as a nucleotide probe or primer, characterized in that it comprises at least 12 consecutive nucleotides of a nucleic acid as defined above, in particular of a nucleic acid with nucleotide sequences SEQ ID No. 1 to SEQ ID No. 4.
• the invention also relates to a nucleic acid which can be used as a nucleotide probe or primer, characterized in that it consists of a polynucleotide of at least 12 consecutive nucleotides of a nucleic acid according to the invention, quite favorite of a nucleic acid of sequences chosen from the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 4.
• nucleic acid can further be characterized in that it is labeled with a detectable molecule.
• a nucleic acid which can be used as a nucleotide probe or primer for the detection or amplification of a genomic sequence, of the mRNA or of the cDNA of the lsum2A gene can also be characterized in that it is chosen from following sequences: a) the nucleotide sequences hybridizing, under high stringency hybridization conditions, with a nucleic acid of sequence
• the present invention also relates to a method for detecting the presence of a nucleic acid of the lsum2A gene in a sample, said method comprising the steps of:
• the oligonucleotide probe (s) are immobilized on a support.
• the oligonucleotide probes include a detectable marker.
• the invention further relates to a kit or kit for detecting the presence of a nucleic acid according to the invention in a sample, said kit comprising: a) one or more nucleotide probes as described above; b) where appropriate, the reagents necessary for the hybridization reaction.
• the detection kit or kit is characterized in that the probe or probes are immobilized on a support.
• the detection kit or kit is characterized in that the oligonucleotide probes comprise a detectable marker.
• such a kit will comprise a plurality of oligonucleotide probes according to the invention which will be used to detect target sequences of interest of the lsum2A gene or alternatively to detect mutations in coding regions or non-coding regions of the lsum2A gene, more particularly nucleic acids of sequence SEQ ID No. 1 to SEQ ID No. 4 or nucleic acids of complementary sequence.
• target sequence within the meaning of the invention means a nucleotide sequence included in a nucleic acid, said nucleotide sequence hybridizing, under the hybridization conditions specified in the description, with a probe or a nucleotide primer of the invention .
• a target sequence can for example be a sequence included in a nucleic acid regulating the lsum2A gene or else a sequence included in a genomic coding region or of the cDNA of this gene.
• nucleotide primers according to the invention can be used to amplify any nucleotide fragment (gDNA,
• CDNA, mRNA of the lsum2A gene, and more particularly all or part of a nucleic acid of sequence SEQ ID No. 1 to SEQ ID No. 4, or else a fragment or a variant of these sequences.
• Another subject of the invention relates to a method for the amplification of a nucleic acid according to the invention, and more particularly of a nucleic acid of sequence SEQ ID No. 1 to SEQ
• said method comprising the steps of: a) contacting the sample in which the presence of the target nucleic acid is suspected with a pair of nucleotide primers whose hybridization position is localized respectively on the 5 ′ side and on the 3 ′ side of the target nucleic acid region of the gene lsum2A whose amplification is sought, in the presence of the reagents necessary for the amplification reaction; and b) detection of the optionally amplified nucleic acid.
• the subject of the invention is also a kit or kit for the amplification of a nucleic acid according to the invention, and more particularly all or part of a nucleic acid of sequences SEQ ID No. 1 to 4, said kit or kit comprising: a) a pair of nucleotide primers according to the invention, the hybridization position of which is located respectively on the 5 ′ and 3 ′ side of the target nucleic acid of the lsum2A gene, the amplification of which is sought ; b) where appropriate, the reagents necessary for the amplification reaction.
• primers according to the invention comprise all or part of a polynucleotide chosen from the nucleotide sequences SEQ ID Nos. 10 to 13 and 17 to 23.
• said cell carries at least one non-functional isum2A allele. Most preferably, said cell carries the two non-functional isum2A alleles.
• Nucleic acid constructs have been developed according to the invention making it possible to obtain a controlled expression of at least one functional copy of a nucleic acid coding for an ISUM2A polypeptide in a cellular host, particularly in a plant cell.
• the subject of the invention is also a nucleic acid comprising a polynucleotide coding for an ISUM2A polypeptide as defined in the present description, or for a fragment of this polypeptide, said nucleic acid further comprising a regulatory polynucleotide capable of regulating the transcription or translation of the polynucleotide encoding the ISUM2A polypeptide, or the fragment of this polypeptide.
• nucleic acid of sequence complementary to the nucleic acid as defined above.
• a nucleic acid construct comprising a polynucleotide coding for an ISUM2A polypeptide as well as a regulatory polynucleotide is also designated “expression cassette” in the present description.
• an expression cassette according to the invention will comprise at least one polynucleotide coding for an ISUM2A polypeptide, the other functional elements allowing the expression of the ISUM2A polypeptide can be carried by a vector in which the expression cassette can be inserted.
• An expression cassette according to the invention can also contain, in a nonlimiting manner, in addition to a regulatory polynucleotide, also other functional elements such as leader sequences or a terminator sequence or alternatively initiation and termination sequences of the transcription.
• the above regulatory nucleic acids can be used to overexpress the polynucleotide coding for the ISUM2A polypeptide or a fragment of the ISUM2A polypeptide, in particular when obtaining plants having seeds with large oil-rich embryos are sought.
• the invention also relates to a nucleic acid coding for an ISUM2A polypeptide or for a fragment of an ISUM2A polypeptide as defined above and comprising a regulatory polynucleotide regulating the transcription and / or translation of the coding sequence, in which the regulatory polynucleotide allows a high level of transcription of the corresponding mRNA and / or a high level of translation of the corresponding polypeptide in the host organism, including host cell or plant, in which it is expressed.
• the invention also relates to the use of a nucleic acid as defined above, of a recombinant vector comprising this nucleic acid or also of a host cell transfected or transformed with this nucleic acid for obtaining '' a transformed plant capable of producing oil-rich seeds.
• a controlled expression of the polynucleotide coding for the ISUM2A polypeptide is sought, especially when the production of seeds with development of affected germ is sought.
• a defect in the expression of the isum2A gene causes the production of seeds without germ, these seeds being not fertile and not allowing the multiplication of the plants mutated in the isum2A gene.
• isum2A gene appears to be necessary for normal plant growth before pollination.
• Pollination is the time when mature pollen comes into contact with a receptive silk.
• obtaining a production of seeds with development of a germ affected by a plant involves: a) normal expression of at least one functional copy of a nucleic acid coding for an ISUM2A polypeptide from the germination stage from seed to pollination of the plant; and b) an absence of the transcription or of the translation of this nucleic acid as soon as the plant is pollinated, in order to produce seeds affected in the development of the germ.
• nucleic acid constructs in which the polynucleotide coding for a polypeptide
• ISUM2A is placed under the control of a regulatory polynucleotide whose activity can be controlled over time.
• the invention therefore also relates to a nucleic acid comprising a polynucleotide coding for an ISUM2A polypeptide chosen from sequences having at least 95% amino acid identity with the sequences SEQ ID No. 5 and SEQ ID No. 6 and which also comprises a regulatory polynucleotide sensitive to the action, direct or indirect, of an inducing signal, also designated as an inducible regulatory polynucleotide.
• the regulatory polynucleotide is a “repressor” polynucleotide of transcription or translation.
• repressor regulatory polynucleotide is meant, according to the invention, a regulatory sequence whose constitutive activity can be blocked by an external signal.
• an external signal may be the absence of binding of a transcription factor recognized by the repressor regulatory polynucleotide. The absence of binding of the transcription factor can be induced under the effect of the repressor inducing signal to which the repressor regulatory polynucleotide is sensitive.
• the expression of the sequence coding for an ISUM2A polypeptide is constitutive in the selected cellular host, in the absence of the repressor inducing signal to which the repressor regulatory polynucleotide is directly or indirectly sensitive.
• the contact of the cellular host with the repressor inducing signal has the effect, thanks to a direct or indirect action on the repressor regulatory polynucleotide, to inhibit and / or block the expression of the polynucleotide coding for the polypeptide ISUM2A.
• the regulatory polynucleotide is a polynucleotide which activates transcription or translation.
• the regulatory polynucleotide activating transcription or translation is sensitive, directly or indirectly, to the action of an activating inducing signal. It is then a polynucleotide "inducible activator" within the meaning of the invention.
• a regulatory polynucleotide of the “inducible activator” type is a regulatory sequence which is only activated in the presence of an external signal.
• an external signal can be the binding of a transcription factor, the binding of a transcription factor being able to be induced under the effect of the activating inducing signal to which the regulatory polynucleotide is directly or indirectly sensitive.
• the expression of the polynucleotide coding for an ISUM2A polypeptide according to the invention can be induced by bringing the transformed cellular host into contact with the activating inducing signal to which the polynucleotide activator regulator is directly or indirectly sensitive.
• Table 3 Examples of ISUM2A inducible expression systems.
• a isum 2A7isum2A " homozygous for a non-functional copy of the isum2A gene, for example a mutation.
• b lsum 2A + / lsum2A + homozygote carrying two functional copies of the isum2A gene.
• the inducible expression system I of Table 3 constitutes an illustration of a construction of nucleic acids in which the regulatory polynucleotide is an “inducible activator” polynucleotide of transcription and / or translation, according to the second embodiment defined above.
• the expression system I comprises: (i) a first expression cassette comprising a nucleic acid coding for an ISUM2A polypeptide placed under the control of a promoter whose activity is induced only in the presence of an activating compound, when this activating compound, generally a transcription factor, is linked to an inducing compound. (ii) a second expression cassette comprising a nucleic acid coding for the activating compound, for example the transcription factor above, placed under the control of a promoter allowing the constitutive expression of the activating compound.
• the transcription or translation of the sequence coding for an ISUM2A polypeptide is induced only when the activating compound is complexed with the inducing compound.
• the complex between the activating compound and the inducing compound binds to the promoter controlling the expression of the nucleic acid coding for an ISUM2A polypeptide, and activates the expression of the latter.
• An example of the system I is illustrated in example 3, in which the system comprises:
• a nucleic acid coding for an ISUM2A polypeptide placed under the control of a promoter containing the UAS sequence, which is recognized by the activating compound GVG.
• the activator compound GVG is a fusion protein between the DNA binding domain of the GAL4 protein, the activator domain of the VP16 gene and the rat glucocorticoid receptor (GR).
• the GVG fusion protein is expressed constitutively in the plant, but does not activate the promoter controlling the expression of the ISUM2A polypeptide, in the absence of a glucocorticoid.
• the glucocorticoid is fixed on the fusion protein GVG, and the complex thus formed between the activating inducing compound (glucocorticoid) and the activating compound (the protein of GVG fusion) will bind to the UAS sequence of the promoter controlling the expression of an ISUM2A polypeptide, which activates the promoter containing the UAS sequence and induces the synthesis of the ISUM2A polypeptide.
• the glucocorticoid constitutes the inducing compound.
• the activating inducing signal is the binding of the activating compound / inducing compound complex (GVG / glucocorticoid fusion polypeptide) to the activating regulatory polynucleotide (promoter containing the UAS sequence).
• the expression system I will preferably be used in a plant cell host or in a plant in which the two copies of the isum2A gene are non-functional, for example in which the two copies of the isum2A gene are mutated.
• the inducible expression system II is an illustration of the embodiment of a nucleic acid construction according to the invention, according to the first embodiment described above, in which the polynucleotide coding for an ISUM2A polypeptide is placed under the control of a "repressor" regulatory polynucleotide.
• the expression system contains two expression cassettes, respectively:
• a first expression cassette comprising a nucleic acid coding for an ISUM2A polypeptide, placed under the control of a regulatory region whose activity is constitutive because it is induced in the presence of an activating compound produced so constitutive;
• a second expression cassette comprising a nucleic acid coding for the activating compound active on the regulatory region of the expression cassette (i) above, said nucleic acid coding for the activator compound being placed under the control of a constitutive promoter, preferably a strong constitutive promoter, or else a promoter active specifically in the cells of the seed embryo.
• the activating compound loses its function of activating the regulatory region controlling the expression of the nucleic acid coding for an ISUM2A polypeptide, when this activating compound is placed in the presence of a specific iigand, which is here designated repressor inducing compound.
• a specific iigand which is here designated repressor inducing compound.
• the activating compound when the activating compound is placed in the presence of the repressor inducing compound with which it binds, the activating compound is deactivated and no longer binds to the regulatory region controlling the expression of the nucleic acid coding for an ISUM2A polypeptide. In this situation, the ISUM2A polypeptide is no longer expressed.
• Example 2 illustrates a particular embodiment of a system II as defined above.
• the system II presented in Example 2 comprises respectively:
• a first expression cassette comprising a nucleic acid coding for the activating compound tTA, placed under the control of a strong constitutive promoter, the promoter of the rice actin 1 gene;
• a second expression cassette comprising a nucleic acid coding for an ISUM2A polypeptide placed under the control of a promoter containing a TRE motif, the promoter being activated when the tTA activator binds to the TRE element.
• the activating compound tTA is produced constitutively and activates the promoter controlling the sequence coding for an ISUM2A polypeptide.
• the tTA activator is deactivated and no longer binds to the TRE motif of the promoter controlling the nucleic acid encoding a 1SUM2A polypeptide.
• the ISUM2A polypeptide is no longer expressed.
• the repressor inducing signal is the absence of binding of the activator compound / repressor inducer compound complex (tTA tetracycline activator) on the repressor regulatory polynucleotide (promoter containing the TRE motif).
• an expression system II as defined above will be used in a plant cell host or in a plant for which the two copies of the isum2A gene are mutated or inactivated.
• the expression system III which is summarized in Table 3, is very similar, with regard to the elements for regulating the expression of the expression cassettes which it contains, to the expression system I above.
• an antisense polynucleotide capable of inhibiting the production of an ISUM2A polypeptide in a plant comprising at least one functional copy of the lsum2A gene, preferably in a plant comprising both copies of the functional isum2A gene.
• the expression system III comprises two expression cassettes, respectively:
• an expression cassette comprising a nucleic acid coding for the activator compound, this nucleic acid being placed under the control of a promoter constituting or specific for the embryo, preferably a strong promoter.
• a promoter constituting or specific for the embryo, preferably a strong promoter.
• the activated activating compound binds to the promoter controlling the expression of the antisense polynucleotide isum2A which activates the expression of the antisense polynucleotide resulting in inhibition of translation of the isum2A gene.
• the expression system III Thanks to the expression system III, the synthesis of the ISUM2A polypeptide is therefore inhibited in the presence of the inducing compound activating the activating compound.
• Such an expression system makes it possible to precisely control the moment when it is desired to block the synthesis of the ISUM2A polypeptide, by bringing the deactivated activating compound into contact with the inducing compound activating the activating compound, for example from the start of pollination.
• An illustrative example of an expression system III can be directly derived from that described in Example 3, when the nucleic acid coding for an ISUM2A polypeptide is replaced by a nucleic acid coding for an antisense polynucleotide inhibiting the translation of the ISUM2A polypeptide or any other method of inhibiting endogenous genes using a transgene and known to those skilled in the art (co-suppression, ribozyme, double-stranded RNA, etc.).
• the inducible expression systems of an ISUM2A polypeptide comprise several expression cassettes which can either be included in a single expression vector, or on the contrary in distinct expression vectors.
• An inducible expression system such as systems I, Il and
• the selection marker gene is carried by the vector comprising the expression cassette or cassettes constituting the inducible expression system.
• the selection marker gene is carried by a vector distinct from a vector comprising the expression cassette or cassettes constituting the inducible expression system.
• an inducible activating regulator polynucleotide chosen from those described below will be used.
• the regulatory sequence capable of controlling the nucleic acid coding for an ISUM2A polypeptide according to the invention can be a regulatory sequence inducible by a particular metabolite, such as:
• - a regulatory sequence inducible by tetracycline such as that sold by the company CLONTECH. - a promoter sequence inducible by a pathogen or by a metabolite produced by a pathogen.
• the nucleic acid allowing the synthesis of the ISUM2A polypeptide can also contain one or more other sequences containing regulatory signals for the expression of the region coding for ISUM2A, or alternatively be placed under the control of such regulatory sequences.
• the other sequences containing regulatory signals include 5 ′ untranslated “leader” sequences.
• leader sequences.
• Such sequences can increase the translation of mRNA encoding ISUM2A.
• EMCV ElectrophaloMyoCarditis VIRUS 5 'non coding region
• the nucleic acid allowing the synthesis of the ISUM2A polypeptide, or the vector into which this nucleic acid is inserted can also comprise so-called "terminator" sequences.
• the expression cassette according to the invention can comprise a polynucleotide coding for an ISUM2A polypeptide fused to a gene regulatory sequence of fragment GR type receptor for glucocorticoids (Aoyma et al. 1997), said polynucleotide being placed under the control of a promoter sequence of the ISUM2 native promoter or constitutive promoter type. In the presence of the hormone, the resulting hybrid protein is no longer retained in the cytoplasm and can therefore enter the chloroplast and integrate into the ribosome.
• a nucleic acid allowing the synthesis of the ISUM2A polypeptide can be inserted into an appropriate vector.
• vector in the sense of the present invention, is meant a circular or linear DNA or RNA molecule which is either in single strand or double strand form.
• a recombinant vector according to the invention is preferably an expression vector, or more specifically an insertion vector, a transformation vector or an integration vector.
• It may especially be a vector of bacterial or viral origin.
• the nucleic acid allowing the synthesis of the ISUM2A polypeptide is placed under the control of one or more sequences containing signals for regulating its expression in the plant considered, that is to say that the regulatory signals are all contained in the nucleic acid encoding ISUM2A, as is the case in the nucleic acid constructs described in the previous section, either that one, more of them, or all of the regulatory signals are contained in the recipient vector into which the nucleic acid encoding ISUM2A has been inserted.
• a recombinant vector according to the invention advantageously comprises appropriate sequences for initiating and stopping transcription.
• the recombinant vectors according to the invention may include one or more origins of functional replication in the host cells in which their expression is sought, as well as, where appropriate, nucleotide selection marker sequences.
• the recombinant vectors according to the invention can also include one or more of the expression regulation signals as defined above in the description.
• the preferred bacterial vectors according to the invention are for example the vectors pBR322 (ATCC No. 37 017) or also the vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, Wl, United States ). Mention may also be made of other commercial vectors such as the vectors pQE70, pQE60, pQE9 (Quiagen), psiX174, pBluescript SA, pNH8A, pMH16A, pMH18A, pMH46A, pWLNEO, pSV2CAT, pOG44, pXTI and pSG (Stratagene).
• They may also be vectors of the Baculovirus type such as the vector pVL1392 / 1393 (Pharmingen) used to transfect the cells of the line Sf9 (ATCC No. CRL 1711) derived from Spodoptera frugiperda.
• vectors of the Baculovirus type such as the vector pVL1392 / 1393 (Pharmingen) used to transfect the cells of the line Sf9 (ATCC No. CRL 1711) derived from Spodoptera frugiperda.
• vectors pRDP ⁇ and pRDP4 described in Example 2 are also included in the invention.
• nucleic acid coding for an ISUM2A polypeptide according to the invention placed under the control of an appropriate regulatory sequence, the nucleic acids or the recombinant vectors defined in the present description must be introduced into a host cell.
• the introduction of the polynucleotides according to the invention into a host cell can be carried out in vitro, according to techniques well known to those skilled in the art.
• Such a transformed host cell is preferably of bacterial, fungal or vegetable origin.
• bacteria cells of different strains of Escherichia coli or of Agrobacterium tumefaciens can in particular be used.
• the transformed host cell is a plant cell or even a plant protoplast.
• cells capable of being transformed according to the process of the invention mention may be made, by way of examples, of cells from plants of large crops (corn, wheat, rapeseed, sunflower, peas, soybeans, barley, etc.).
• plants known for contain large reserves protein, carbohydrate and lipid), especially cereal plants or oil plants.
• the hybrid plants obtained by crossing plants according to the invention also form part of the invention.
• it is a cell or a protoplast of a cereal plant.
• the cell or protoplast is preferably native to corn, wheat, barley, sorghum, millet, rye or rice.
• the subject of the invention is also the use of a nucleic acid comprising a polynucleotide coding for an ISUM2A polypeptide, where appropriate in the form of a nucleic acid construct as defined above, for manufacturing a transformed plant capable of producing seeds with development of the affected germ.
• the invention also relates to the use of a recombinant vector as defined in the present description for manufacturing a transformed plant capable of producing seeds with development of the affected germ.
• the invention also relates to the use of a cellular host transformed with a nucleic acid comprising a polynucleotide coding for an ISUM2A polypeptide, where appropriate in the form of a nucleic acid construct or expression cassette as defined above. above, to make a transformed plant capable of producing seeds with development of the affected germ.
• the invention also relates to a transformed plant comprising a plurality of host cells as defined above.
• the invention also relates to a transformed multicellular plant organism, characterized in that it comprises a transformed host cell or a plurality of host cells transformed by an acid.
• nucleic acid comprising a polynucleotide encoding the ISUM2A polypeptide as defined in the present description or also by a recombinant vector comprising such a nucleic acid.
• the subject of the invention is also a transformed plant comprising, in a form artificially integrated into its genome, a nucleic acid allowing the synthesis of the ISUM2-A polypeptide, as defined in the present description.
• the transformed plant may contain a plurality of copies of a nucleic acid encoding the ISUM2A polypeptide, in situations in which overexpression of the ISUM2A polypeptide is sought.
• An overexpression of the ISUM2A polypeptide is sought in particular when it is desired to obtain plants producing seeds whose germ has a significantly larger size than in "wild" plants and which is enriched in oil.
• the overexpression of the ISUM2A polypeptide can be obtained by transforming a host cell or a plant with a nucleic acid coding for the ISUM2A polypeptide and in which the polynucleotide comprising the open reading frame is placed under the control of an acid. regulatory nucleic acid allowing a high level of transcription of the corresponding mRNA or a high level of translation of the ISUM2A polypeptide in the host cell or in the plant.
• the invention therefore also relates to a transformed plane as defined above, the seeds of which are rich in oil.
• It also relates to a process for obtaining such a transformed plant.
• field crops preferably corn, wheat, rapeseed, sunflower, peas, soybeans and barley.
• the preferred plants according to the invention are those of which the seeds have a high starch content or a high amount of starch and most preferably corn, wheat, barley, sorghum, millet, rye or rice.
• Hybrid plants obtained by crossing transformed plants according to the invention also form part of the invention.
• the invention also relates to any part of a transformed plant as defined in the present description, such as the root, but also the aerial parts such as the stem, the leaf, the flower and especially the seed.
• the subject of the invention is also a seed or a seed of a plant produced by a transformed plant as defined above.
• a transformed seed or such a transformed grain comprises one or more cells comprising in their genome one or more copies of a nucleic acid allowing the synthesis of the ISUM2A polypeptide, if necessary in a controlled and inducible manner.
• the seeds or seeds of plants are enriched in oil.
• the invention therefore also relates to seeds and grains enriched in oil, prepared or obtained from a transformed plant overexpressing the ISUM2A polypeptide.
• Oil rich seeds are used for seed preparation or oil enriched seed meal for use in agriculture and the food industry.
• a transformed plant it is sought to express in a controlled manner the ISUM2A polypeptide, which implies that the transformed plant does not contain, as a functional copy of a polynucleotide encoding the polypeptide ISUM2A, only the copy or copies which have been artificially introduced into their cells, and preferably into their genome, while the sequences of the isum2A gene found naturally in the wild plant carry at least one mutation causing a defect in the expression of the isum2A gene .
• Such plants mutated in the isum2A gene are, for example, the mutant plants G2422 or the mutant plants emb * - 8516 described by HECKEL et al. (1999).
• a person skilled in the art can, thanks to the invention, produce other plants mutated in the isum2A gene, for example by random insertion of the Mutator transposon in a population of plants of wild phenotype (lsum2A7lsum2A + ), then by detecting in the mutants obtained, those of these mutants which no longer express the isum2A gene, for example using the probes or nucleotide primers described in the examples.
• the transformed plant according to the invention is characterized in that it derives from a plant in which the two copies of the isum2A gene each carry at least one mutation causing a defect in its expression, at the transcriptional level or translational.
• a second preferred embodiment according to the invention it is sought to inhibit in a controlled manner the synthesis of the ISUM2A polypeptide, for example through the expression of an antisense polynucleotide.
• the transformed plant comprises at least one functional copy of the isum2A gene and preferably the two copies of the isum2A gene are functional.
• the invention also relates to a process for obtaining a transformed plant capable of producing seeds affected in the development of the germ, characterized in that it comprises the following stages: a) transformation of at least one cell vegetable; - with a nucleic acid comprising a polynucleotide coding for an ISUM2A polypeptide chosen from sequences having at least 95% amino acid identity with the sequences SEQ ID No. 5 and SEQ ID No.
• nucleic acid also comprising a polynucleotide regulator as defined in the present description; or - by a recombinant vector comprising such a nucleic acid; b) selection of the cells transformed in step a) having integrated into their genome at least one copy of a nucleic acid coding for the polypeptide ISUM2A. c) regeneration of a transformed plant from the transformed cells obtained in step b);
• seeds affected in the development of the germ is meant according to the invention seeds whose development of the germ is “abnormal”, that is to say significantly different from the germ of a seed of a plant called “wild” .
• the development of the germ can be affected so that its size is significantly larger than that of the germ found in the seeds of “wild” plants not affected in the expression of the isum2A gene.
• a significantly larger size of the germ can be obtained by overexpressing the ISUM2A polypeptide, for example either by introducing a plurality of copies of a nucleic acid encoding this polypeptide into a host cell or in a plant, or by placing a copy of the isum2A gene or its cDNA under the control of a regulatory polynucleotide allowing the overexpression of the ISUM2A polypeptide in the host cell or in the plant.
• the invention also relates to a particular embodiment of the above process for obtaining a transformed plant capable of producing seeds with development of the affected germ in which at least one plant cell is transformed in step a ) by Agrobacterium tumefaciens containing: - a nucleic acid comprising a polynucleotide coding for an ISUM2A polypeptide chosen from sequences having at least 95% amino acid identity with the sequences SEQ ID No. 5 and SEQ ID No. 6 and comprising a regulatory polynucleotide as defined in the present description; or - a recombinant vector comprising such a nucleic acid;
• Each of the methods for obtaining a transformed plant according to the invention can comprise the following additional steps: d) crossing of a plant selected in step c) with a heterozygous plant comprising a functional copy of the isum2A gene and a copy isum2A gene inactive; e) selection of the plants resulting from the crossing of step d) which are homozygous and carry two inactive copies of the isum2A gene.
• the regulatory polynucleotide is sensitive to the action of an inducing signal; it is also said that the regulatory polynucleotide is inducible.
• the inducible regulatory polynucleotide consists of a regulatory "repressor" polynucleotide as defined in the present description.
• the inducible regulatory polynucleotide is a regulatory polynucleotide "activator" of transcription or translation as defined in the present description.
• the present invention also relates to a transformed plant, as obtained by one of the production methods defined above.
• the invention also relates to a hybrid transgenic plant obtained by crossing a transformed plant as defined above.
• the invention also relates to a part of a plant transformed according to the invention.
• the subject of the invention is also a process for obtaining grains of plants with development of the affected germ, characterized in that it comprises the following stages: a) cultivating, until pollination, a plant of which the two copies of the isum2A gene carry at least one mutation causing a defect in the production of the ISUM2A polypeptide and in the genome of which a nucleic acid has been artificially introduced comprising:
• the development of the germ can be affected in such a way that its size is significantly reduced compared to that of seeds from wild plants, or even nonexistent, as has been shown when the plant has a non-functional isum2A gene or when we introduce a nucleic acid coding for the ISUM2A polypeptide under the control of a regulatory polynucleotide making it possible to block transcription or translation in a controlled manner in order to affect the development of the germ.
• the present invention also relates to a process for obtaining grains of plants with development of the affected germ, characterized in that it comprises the following stages: a) cultivating, until pollination, a plant of which the two copies of the isum2A gene each carry at least one mutation causing a defect in the production of the ISUM2A polypeptide and in the genome of which a nucleic acid has been artificially introduced comprising:
• the invention also relates to a seed with development of the affected germ as it is obtained according to one of the methods defined above.
• the invention also relates to a developing seed of the affected germ, characterized in that each of its constituent cells comprises, in a form artificially integrated into their genome, a nucleic acid comprising: - a polynucleotide coding for an ISUM2A polypeptide chosen from sequences having at least 95% amino acid identity with the sequences SEQ ID No. 5 and SEQ ID No. 6; and
• the regulatory polynucleotide consists of an inducible regulatory polynucleotide of the repressor type.
• the regulatory polynucleotide consists of a regulatory polynucleotide of the inducible activator type. Also part of the invention is any transformation product of a seed as defined in the present description, in particular a seed or an oil.
• the transformation product is a starch.
• starch from a seed developing the affected germ according to the invention the skilled person will advantageously use the techniques described in the book "Handbuch der Starke” (vol. I; Max Ullmann ( ed.), Paul Varey Verlag, Berlin) or in the article by Morrison and Karkalas (Methods in Plant Biochemistry, 1990, vol. 2: 323-352, Académie Press Ltd; London).
• the starch obtained from seeds with development of the affected germ according to the invention can be used by the food industry, the pharmaceutical industry, the paper industry or even in the microbiological field, where it can be used. as a nutrient substrate.
• the transformation of plant cells can be carried out by techniques known to those skilled in the art.
• direct gene transfer methods such as direct microinjection into plant embryoids (NEUHAUS et al, 1987), vacuum infiltration (BECHTOLD et al., 1993) or electroporation (CHUPEAU et al , 1989) or direct precipitation by means of PEG (SCHOCHER et al, 1986) or the bombardment by cannon of particles covered with the plasmid DNA of interest (FROMM M. e
• the plant cells are transformed by a vector according to the invention, said cell host being capable of infecting said plant cells by allowing the integration into the genome of these latter, sequences Nucleotides of interest initially contained in the DNA of the above-mentioned vector.
• the above-mentioned cell host used is Agrobacterium tumefaciens, in particular according to the method described in the article by AN et al, (1986), or else Agrobacterium rhizogenes, in particular according to the method described in the article by GUERCHE et al, (1987) or in PCT application No. WO OO 22.148.
• the transformation of plant cells can be carried out by the transfer of the T region of the extrachromosomal circular plasmid inducing Ti tumors of Agrobacterium tumefaciens, using a binary system (WATSON et al. 1994).
• a binary system WATSON et al. 1994.
• two vectors are constructed. In one of these vectors, the T region has been deleted, with the exception of the right and left borders, between them the gene of interest, as well as a marker gene are inserted to allow selection in cells of plants.
• the other partner of the binary system is a helper Ti plasmid, a modified plasmid which no longer has a T region but still contains the vir virulence genes, necessary for the transformation of the plant cell.
• the method described by ISHIDA et al. Can be used. (1996) for the transformation of Monocotyledons.
• the transformation is carried out according to the method described by FINER et al. (1992) using the tungsten or gold particle gun.
• a person skilled in the art is capable of implementing numerous methods of the state of the art in order to obtain plants transformed with a nucleic acid allowing the synthesis of the ISUM2A polypeptide.
• transgenic plant according to the invention can be obtained by biolistic techniques such as those described by FINER et al. (1992) or those described by VAIN et al. (1993).
• the isum2A gene codes for a polypeptide having a length of 143 amino acids, for which at least two variant polypeptides have been observed.
• the first variant polypeptide encoded by the isum2A gene has the amino acid sequence SEQ ID No. 5 and is encoded by the isum2A gene present in the genome of the corn plant designated HD5x HD7.
• the second variant ISUM2A polypeptide is encoded by the isum2A gene present in the genome of the corn plant designated A188.
• the ISUM2A polypeptides of sequences SEQ ID No 5 and SEQ ID No 6 differ only by the substitution of an amino acid residue, the polypeptide of sequence SEQ ID No 5 having a residue glycine at position 89 and the polypeptide of sequence SEQ ID No. 6 having an amino acid residue asparagine at this position.
• ISUM2A leads in all cases to the expression of a wild phenotype in the plant, said plant producing mature and fertile grains comprising a fully developed embryo.
• No. 5 has a calculated molecular weight of 15,112 daltons, a calculated isoelectric point of 11.75 and a charge at pH 7 of 28.19.
• the ISUM2A polypeptide of sequence SEQ ID No. 5 has 40 charged amino acid residues (R, K, H, Y, C, D, E), 3 acidic amino acid residues (D, E), 31 residues d basic amino acids (K, R), 31 polar amino acid residues (N, C, Q, S, T, Y) and 55 hydrophobic amino acid residues (A, I, L, F, W, V ).
• the ISUM2A polypeptide of sequence SEQ ID No. 6 has characteristics very similar to those described below above for the ISUM2A polypeptide of sequence SEQ ID No. 5.
• a subject of the invention is therefore also the polypeptide comprising the amino acid sequence SEQ ID No. 5 or SEQ ID No. 6 as well as a polypeptide having at least 95% amino acid identity with the sequence SEQ ID N ° 5 or SEQ ID No. 6, or a fragment or variant thereof.
• a fragment of an ISUM2A polypeptide according to the invention comprises at least 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 135 or 140 consecutive amino acids of a polypeptide of sequence SEQ ID No. 5 or SEQ ID No. 6.
• polypeptide comprising
• the invention also relates to a polypeptide comprising an amino acid sequence having at least 95% amino acid identity with the sequence of an ISUM2A polypeptide of sequence
• polypeptides according to the present invention are in an isolated or purified form.
• Another subject of the invention consists of a polypeptide comprising amino acid modifications of 1, 2, 3, 4 or 5 substitutions, additions or deletions of an amino acid with respect to the amino acid sequence of a polypeptide of sequence SEQ ID No. 5 or
• SEQ ID N ° 6 or a fragment or a variant thereof.
• a polypeptide according to the invention can be obtained by genetic recombination according to techniques well known to those skilled in the art, for example techniques described in AUSUBEL
• a polypeptide according to the invention can also be prepared by conventional techniques of chemical synthesis, either in homogeneous solution or in solid phase.
• a polypeptide according to the invention may be prepared by the technique in homogeneous solution described by HOUBEN WEIL (1974) or also by the solid phase synthesis technique described by MERRIFIELD (1965a; 1965b).
• variant polypeptides of a polypeptide according to the invention retain their ability to be recognized by antibodies directed against the polypeptides of sequences SEQ ID No. 5 or 6.
• a polypeptide encoded by the lsum2A gene according to the invention such as a polypeptide of amino acid sequence SEQ ID No. 5 or 6, or a variant or a peptide fragment of the latter is useful in particular for the preparation of antibodies intended for the detection of presence and / or expression of a polypeptide of sequences SEQ ID No. 5 or 6 or of a peptide fragment of the latter in a sample.
• antibodies directed against these polypeptides are used to quantify the synthesis of a polypeptide of sequences SEQ ID No. 5 or 6, for example in cells of a plant, and thus determine the development of the embryo.
• antibody within the meaning of the present invention, is meant in particular polyclonal or monoclonal antibodies or fragments (for example the fragments F (ab) ' 2 , F (ab)) or any polypeptide comprising a domain of the antibody initial recognizing the polypeptide or the fragment of target polypeptide according to the invention.
• Monoclonal antibodies can be prepared from hybridomas using the technique described by KOHLER and MILSTEIN (1975)
• the present invention also relates to antibodies directed against a polypeptide as described above or a fragment or a variant thereof, as produced in the trioma technique or also the hybridoma technique described by KOZBOR et al. (1983).
• the invention also relates to fragments of single chain Fv antibody (ScFv) as described in US Patent No. 4,946,778 or by MARTINEAU et al. (1998).
• the antibodies according to the invention also comprise fragments of antibodies obtained using phage libraries as described by RIDDER et al. (1995) or humanized antibodies as described by REINMANN et al. (1997) and LEGER et al. (1997).
• the antibody preparations according to the invention are useful in immunological detection tests intended for the identification of the presence and / or of the quantity of a polypeptide of sequences SEQ ID No. 5 or 6, or of a fragment peptide thereof, present in a sample.
• an antibody according to the invention may also comprise an isotopic or non-isotopic detectable marker, for example fluorescent, or else be coupled to a molecule such as biotin, according to techniques well known to those skilled in the art.
• the subject of the invention is also a method for detecting the presence of a polypeptide in accordance with the invention in a sample, said method comprising the steps of: a) bringing the sample to be tested into contact with an antibody such as described above; b) detecting the antigen / antibody complex formed.
• the invention also relates to a kit or diagnostic kit for detecting the presence of a polypeptide according to the invention in a sample, said kit comprising: a) an antibody as defined above; b) where appropriate, one or more reagents necessary for the detection of the antigen / antibody complex formed.
• Another subject of the invention consists in the use of a nucleic acid or an allelic variant of a nucleic acid as defined above in selection programs for obtaining plants with an embryo modified in size. and / or development influencing the starch and / or oil content.
• the invention also relates to a method for selecting plants with an embryo modified in size and / or in development, comprising the steps of: a) genotyping plants (individuals) by means of nucleotide probes or primers obtained from nucleic acids as defined previously or variants of these nucleic acids; b) select, among these plants (individuals), those which include a high frequency of favorable alleles associated with the size and / or development of the embryo.
• FIG. 1 illustrates the genomic nucleotide sequence of the isum2A gene present in the corn plant designated HD5 x HD7 referenced as the sequence SEQ ID No. 1 of the sequence listing.
• the three exons of the isum2A gene are represented in the form of boxes, under the corresponding nucleotide sequence.
• the different sites of recognition for restriction endonucleases are indicated, above the corresponding nucleotide sequence.
• sequences derived from the three exons of the gene are represented by boxes located under the corresponding nucleotide sequence.
• the deduced amino acid sequence is shown below the boxes representing the exons.
• the box designated “RL35 domain” corresponds to the part of the sequence of the ISUM2A polypeptide which is homologous with certain proteins of the chloroplast.
• the ISUM2A polypeptide represented in FIG. 2 is referenced as the sequence SEQ ID No. 5 of the sequence listing.
• FIG. 3 illustrates a partial cDNA sequence corresponding to the messenger RNA found in the corn plant designated A188, this nucleotide sequence being referenced as the sequence SEQ ID No. 4 of the sequence listing.
• the deduced sequence of the protein encoded by the cDNA is represented below the corresponding nucleotide sequence, and is referenced as the sequence SEQ ID No. 6 of the sequence listing.
• the boxes located below the peptide sequence represent the three exons derived from the isum2A gene found in the A188 corn line.
• the box designated “RL35 domain” corresponds to the part of the ISUM2A polypeptide having homology with chloroplast proteins.
• FIG. 4 illustrates the structure in exons and introns of the isum2A gene, the boxes representing the exons of the gene.
• the A base of the ATG codon is the nucleotide at position 51 of the first exon and the T base of the TGA codon of translation stop and the nucleotide at position 76 of the third exon.
• the boxes below the gene structure isum2A represent the different genomic DNA clones used in the examples.
• the boxes above the structure of the isum2A gene represent the different cDNA clones used in the examples.
• Figure 5 illustrates a map of plasmid pRDP5.
• FIG. ⁇ illustrates the map of the plasmid pTRE sold by the company CLONTECH LABORATORIES Inc. and the structure of which is accessible in the GENBANK database under the access number U89931.
• FIG. 7 illustrates the map of the plasmid pDM302 described by CAO et al. (1992).
• FIG. 8 illustrates the map of the plasmid pTet-Off sold by the company CLONTECH (catalog reference year 2000: K1620-A).
• Figure 9 illustrates the map of plasmid pRDP4.
• FIG. 10 illustrates the map of the plasmid pTA7001 described by
• Figure 11 illustrates the map of plasmid pRDP2.
• FIG. 13 illustrates the operating principle of the expression system 11 summarized in table 3 and subject of example 2.
• FIG. 13A illustrates the constitutive expression of the ISUM2A polypeptide under the effect of the activation of the promoter containing the TRE sequence by the activator tTA, which is itself constitutively expressed.
• FIG. 13B illustrates the repression of the synthesis of the ISUM2A polypeptide when the activator tTA is brought into contact with tetracycline, which deactivates it and prevents it from activating the promoter containing the TRE sequence.
• FIG. 14 illustrates a diagram of the operation of an expression system I summarized in table 3 and subject of example 3.
• FIG. 14A illustrates the synthesis of the ISUM2A polypeptide when the promoter containing the UAS sequence is activated by the fusion protein GVG, in the presence of a glucocorticoid.
• FIG. 14B illustrates the absence of production of ISUM2A polypeptide in a situation in which the GVG fusion protein is produced in the absence of a glucocorticoid and does not activate the UAS sequence of the promoter controlling the expression of the isum2A gene.
• EXAMPLES EXAMPLE 1: Cloning of the genomic sequence of the isum2A gene.
• the plants were grown either in a climatic chamber, with a lighting period of 16 h (100 Wm-2), a relative humidity of 80% and an alternation of 24 ° C / 19 ° C (day / night), or in greenhouse under the same conditions but without humidity control, either in the open field in Lyon. All plants were pollinated by hand.
• RNAse A The DNA pellet was rinsed with 1 ml 70% ethanol, dried and taken up in 50 ⁇ l TE10.01 (Tris-HCI pH8 10 mM, EDTA pH8 0.1 mM) supplemented with 20 ⁇ g / ml of RNAse A. A4. PCR on genomic DNA
• the genomic DNA was diluted 10 times and 2 ⁇ l were used in a 20 ⁇ l reaction containing each primer 1 ⁇ M, dNTP 100 ⁇ M and 1 u Taq polymerase in the buffer provided with the enzyme (Pharmacia).
• the reaction was carried out in a Perkin Elmer DNA Thermal Cycler 2400 or 9700 PCR amplifier as follows: after denaturing the DNA 2 min at 94 ° C, the samples underwent 30 cycles of 30 s denaturation at 94 ° C / 45 s of hybridization at 60 ° C / 1 min elongation at 72 ° C. All primers had Tm between 60 ° and 62 ° C.
• the plasmid DNA was prepared according to the alkaline lysis protocol described by Sambrook et al. (1989). Digests of genomic DNA, plasmid DNA or phada lambda DNA by restriction enzymes were carried out according to the advice of the manufacturers (Gibco, Boehringer Mannhein, Promega). The digestion or PCR amplification products were separated by agarose gel electrophoresis in TAE buffer (Sambrook et al., 1989).
• the restriction fragments were ligated into the vector pBluescript and the PCR products into pGEM-T-easy, using the T4-DNA ligase following the advice of the supplier (Promega), using 50 ng of vector, and a insert / vector molar ratio of the order of 3/1. These constructs were introduced into the bacterial strain DH5- ⁇ by transformation by thermal shock, according to the protocol of Hanahan (1983).
• Membranes for DNA hybridization were obtained either by transfer of restriction fragments of agarose gels onto Hybond N + membranes (Amersham) by capillary action, in the presence of 0.4 N sodium hydroxide, or by transfer of lysis plaques from lambda phage on Hybond N membranes (Amersham).
• the membranes were prehybridized according to the supplier's advice, for 4 to 12 hours at 65 ° C in the presence of 5 x SSPE (20 x SSPE contains 3.6 M NaCl, 0.2 M sodium phosphate, EDTA pH 7.7 ; 0.02 M). Then they were hybridized for 16 h under the same conditions.
• the probes used for the hybridization were radioactively labeled with 50 ⁇ Ci of ⁇ 32 P-dCTP, using the Ready-To-GO TM DNA labeling beads kit (Amersham). After hybridization, the membranes were subjected to four rinses, at 65 ° C. in 0.1% solutions of SDS and SSC concentrations of 2 x, 1 x, 0.5 x and 0.1 x (SSC 20 x contains 3 M NaCl, 0.3 M Na 3 -citrate) and were exposed to film for autoradiography (X-OMAT Kodak).
• RNAs were extracted from 50 mg of ground tissue in liquid nitrogen with the TRIZOL TM reagent according to the protocol of the supplier (Gibco).
• RNA 6 ⁇ g of purified RNA were “reverse” transcribed by MuLV reverse transcriptase (Gibco) in a total volume of 20 ⁇ l as advised by the supplier, with a polyT primer at a final concentration of 2.5 ⁇ M. After inactivation of the reverse transcriptase by heating at 95 ° C for 5 min, 2 ⁇ l of the reverse transcription reaction were amplified by Taq polymerase as described above.
• sequencing was carried out by the company Génome Express in Grenoble (France). Sequencher 3.1.1 (Gene Codes Corporation) and DNAstar (Laser Gene) software were used to analyze and assemble the sequences obtained.
• an AIMS product of 107 bp was obtained with the primers Mu 16 (SEQ ID No. 8) and adapMsel (SEQ ID No. 9).
• This "AIMS product" (see FIG. 4) contains 70 bp of the flanking sequence between the two primers, which does not show significant homology with the sequences referenced in the databases.
• Genomic walk (Devic et al, 1997) was used on the genomic DNA of genotype A188. With the primers “nested” GW4 (SEQ ID N ° 12) and GW4b (SEQ ID No. 13) and cleavage by Sspl, a product of 244 bp was obtained (clone L223a, FIG. 4), which contained an additional 153 bp of the flanking sequence.
• the 1532 bp product (clone L223c, Fig. 4) contained 1463 bp of additional flanking sequence.
• the sequence of the clone L223c showed homology in the databases with an EST of corn. Two sequences of the same cDNA are found in Genebank: a sequence from the 5 'end in the accession AI001298, and a sequence from the 3' end in the accession AI374506. This EST bears the name Isum2 without explanation of this acronym. The homology was strong but limited to a small part of L223c. A PCR reaction was carried out on genomic DNA with the primers Isum2b (SEQ ID No. 18) and Isum2e (SEQ ID No. 20), which are located on either side of the suspected intron.
• the amplification product was cloned and sequenced.
• the sequence of this clone L157a (fig. 4) showed that the sequence present in the AIMS product, in L223a and in a part of L223c corresponded to an intron of isl2.
• a population of 25,000 plants highly mutagenized with the transposon mutator was screened by PCR for insertion into lsum2. The plants were planted in 10 blocks of 50 x 50 plants.
• DNA pools of 500 rows and 500 columns were analyzed by PCR for the presence of an amplification with the OMuA primer (SEQ ID # 24 in mutator) in combination with a primer in an exon of isl2.
• the four primers Isum2b (SEQ ID N ° 18), Isum2c (SEQ ID N ° 19), Isum2k (SEQ ID N ° 21) and 8516g (SEQ ID N ° 23) specific to lsum2 were tested. Positive results were obtained with the primers Isum2b and Isum2k.
• the plants in question were identified and the amplification was confirmed on individual plant DNA. Then their descendants were analyzed to distinguish somatic insertions (absent in the gametes) from germinative insertions (present in the gametes). Of all the insertions, only one was found in the next generation.
• the sequencing of the PCR amplification products showed that it was an insertion into intron 1 of lum2A at 3 bp upstream of exon 2, found in a
• lsum2A gene For the lsum2A gene, a first genomic sequence was obtained by screening a genomic library of plants of the genotype
• the sequence of the HD5 x HD7 genotype (Fig. 1) emanates from the two subclones L211c1 (Xhol fragment of 9 kb) and L211c2 (Xhol fragment of 6 kb).
• genotype A188 comes from clones L157a (PCR with the primers Isum2b (SEQ ID No 18) and Isum2e (SEQ ID No 20)), L223a (genomic alk, see above) and L223c (genomic walk, see above).
• the partial cDNA sequence of the A188 genotype comes from clones L158a and L254 (fig. 4). The first was obtained by RT-PCR on embryos ⁇ ⁇ T2 ⁇ AP TM av ⁇ c1 ⁇ s ⁇ m ⁇ r ⁇ sTsljm2 ⁇ î (SEQ ID N ° 18) and Isum 2c (SEQ ID N ° 19), the second by RT-PCR on albumen of 7JAP with primers Isum2a (SEQ ID N ° 17) and Isum2l (SEQ ID N ° 22).
• the cDNA sequence of the HD5 x HD7 genotype (SEQ ID No. 3, fig. 2) was obtained from the genomic sequence (SEQ ID No. 1) using the EST Isum2 (above) to determine the limit 5 'of the first exon and the limit 3' of the last exon.
• Construction of a vector comprising a polynucleotide coding for an ISUM2A polypeptide placed under the control of an inducible regulatory polynucleotide of the repressor type.
• This expression system involves the construction of two vectors, respectively:
• the vector pTRE marketed by the CLONTECH Company
• the pTRE vector is cleaved by the BamHI restriction endonuclease, at nucleotides 471 and 483 of the vector, the DNA fragment located between the two aforementioned BamHI sites then being excised.
• Amplification of the isum2A gene described in Example 1 is then carried out, more specifically of the genomic region going from the ATG codon at the end of the reading frame and also comprising a terminator sequence, by PCR amplification.
• the isum2A gene amplification product is ligated into the open pTRE vector in order to construct the pRDP ⁇ vector illustrated in FIG. 5.
• the isum2A gene is placed under the control of the prTRE promoter, which is recognized by the tTA activator.
• the vector pRDP4 contains the gene for the activator tTA under the control of the actinel promoter of rice.
• the pTet-Off vector sold by the company CLONTECH Laboratories Inc. (catalog reference No. K 1620-A, year 2000), which is illustrated in FIG. 8, is used to amplify the sequence of the tTA gene by PCR.
• the amplification product of the tTA gene is ligated at the open SmaI site of the plasmid pDM302 in order to construct the plasmid pRDP4 illustrated in FIG. 9.
• the plasmid pRDP4 comprises the tTA gene under the control of the actinel promoter of rice.
• FIG. 13 A diagram of the operation of the inducible expression system described in the present example is shown in FIG. 13, either in the absence of tetracycline (FIG. 13A), or in the presence of tetracycline (FIG. 13B).
• FIG. 13A A diagram of the operation of the inducible expression system described in the present example is shown in FIG. 13, either in the absence of tetracycline (FIG. 13A), or in the presence of tetracycline (FIG. 13B).
• FIG. 13A A diagram of the operation of the inducible expression system described in the present example is shown in FIG. 13, either in the absence of tetracycline (FIG. 13A), or in the presence of tetracycline (FIG. 13B).
• Tables 5 to 8 A summary of the characteristics of the various vectors used or prepared according to Example 2 is presented in Tables 5 to 8 below.
• Construction of a vector comprising a polynucleotide encoding an ISUM2A polypeptide placed under the control of a regulatory polynucleotide of the inducible activator type.
• the expression system according to Example 3 constitutes an illustration of the expression system I summarized in Table 3, and which is commented on in the description.
• the expression system according to Example 3 comprises two expression cassettes, respectively:
• an expression cassette comprising the isum2A gene placed under the control of a promoter containing the UAS sequence recognized by the GVG activator; and (ii) an expression cassette coding for the GVG activator, placed under the control of the actinel rice promoter, which is a strong and constitutive promoter in monocots.
• the starting vector is the plasmid pTA7001 described by AOYAMA et al. (1997) and which is shown in Figure 10.
• the vector pTA7001 is subjected to digestion with the enzymes Sse8387l and Pmel in order to excise the promoter 35S of the mosaic virus of the cauliflower.
• the rice actinel promoter is amplified by PCR, from the plasmid pDM302 illustrated in FIG. 7.
• the amplification product of the rice actinel promoter is ligated into the vector pTA7001 previously digested, to construct the plasmid pRDP2 illustrated in FIG. 11.
• the starting vector is the vector pRDP2 constructed according to the above protocol.
• the vector pRDP2 is first subjected to digestion using the restriction endonucleases Xhol and Spel.
• the vector pRDP3 contains two expression cassettes, respectively:
• the GVG gene codes for a fusion protein between the DNA binding domain of the GAL4 protein, the activator domain of the VP16 gene and the rat glucocorticoid receptor (GR).
• FIG. 14 A diagram of the functioning of the inducible expression system described in the present example is shown in FIG. 14, either in the presence of a glucocorticoid (FIG. 14A), or in the absence of a glucocorticoid (FIG. 14B).
• the hybrid transcription factor GVG is transported in the nucleus and strongly activates all the promoters containing the UAS sequence ( Figure 14B).
• Tables 9 to 11 below present a summary of the main characteristics of the different vectors used or prepared according to example 3.
• Transformation of a plant in order to obtain a stable expression of the polynucleotide of interest in the transformed plant is necessary to ensure a lasting modification of the corn kernel. Tests are carried out with a construction of vectors described in Examples 2 and 3.
• Target cells are undifferentiated cells in rapid divisions which have retained the ability to regenerate whole plants. This type of cell makes up the embryogenic callus (called type II) of corn. These calluses are obtained from immature Hill genotype embryos according to the method and on the media described by ARMSTRONG (1994) MAIZE HANDBOOK; 1994, M. FREELING, V. WALBOT EDS .; PP 665-671.
• Fragments of such calluses with an area of 10 to 20 mm 2 were placed, 4 h before bombardment, at the rate of 16 fragments per dish in the center of a Petri dish containing a culture medium identical to the initiation medium , supplemented with 0.2 M mannitol + 0.2 M sorbitol.
• the plasmids described in the previous examples and carrying the genes to be introduced are purified on a Qiagen R column according to the manufacturer's instructions. They are then precipitated on tungsten particles (M 10) following the protocol described by KLEIN (1987). The particles thus coated are projected towards the target cells using the cannon and according to the protocol described by J. FINER (1992).
• the callus boxes thus bombarded are then sealed using Scellofrais R and then grown in the dark at 27 ° C.
• the first subculture takes place 24 hours later, then every fortnight for 3 months on medium identical to the initiation medium added with a selective agent.
• Calls are obtained after 3 months or sometimes earlier, calluses whose growth is not inhibited by the selective agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies of the selection gene.
• the frequency of obtaining such calluses is approximately 0.8 cal per bombarded box.
• Agrobacterium tumefaciens according to the protocol described by ISHIDA et al. (1996), in particular from immature embryos 10 days after fertilization. All the media used are referenced in the cited reference.
• the transformation begins with a coculture phase where the immature embryos of the corn plants are brought into contact for at least 5 min with Agrobacterium tumefaciens LBA 4404 containing the superbinary vectors.
• the superbinary plasmid is the result of a homologous recombination between an intermediate vector carrying the T-DNA containing the gene of interest and / or the selection marker derived from the plasmids described in the preceding examples, and the vector pSB1 from Japan.
• Tobacco (EP 672 752) which contains: the virB and virG genes of the plasmid pTiBo542 present in the supervirulent strain A281 of Agrobacterium tumefaciens (ATCC 37349) and a homologous region found in the intermediate vector allowing this homologous recombination.
• the embryos are then placed on LSAs medium for 3 days in the dark and at 25 ° C.
• the second selection step is carried out by transfer of the embryos which have developed on LSD5 medium, on LSD10 medium (phosphinotricin at 10 mg / l) in the presence of cefotaxime, for 3 weeks under the same conditions as above.
• the third selection step consists in excising the type I calluses which remain white (fragments of 1 to 2 mm) and in transferring them 3 weeks in the dark and at 25 ° C on LSD 10 medium in the presence of cefotaxime.
• the regeneration of the seedlings is carried out by excising the type I calluses which have proliferated and by transferring them to LSZ medium in the presence of phosphinotricin at 5 mg / l and cefotaxime for 2 weeks at 22 ° C. and under continuous light.
• the plants which have regenerated are transferred to RM + G2 medium containing 100 mg / l of Augmentin for 2 weeks at 22 ° C. and under continuous illumination for the development stage.
• the plants obtained are then transferred to the phytotron for their acclimatization.
• Biochemical analyzes were carried out on maize seeds obtained after self-fertilization of heterozygous plants carrying the emb8516 mutation.
• the offspring is composed of seeds with wild phenotype, with embryo (either wild homozygotes, or heterozygotes) or with mutant phenotype without embryo (mutant homozygotes). These seeds were sorted manually (visual sorting according to the phenotype) to separate those with a mutant phenotype from those with a wild phenotype.
• EWERS starch (Ewers starch (2)): 3rd European Community directive with JOCE corrigendum of 11/27/80. Determination of starch, polarimetric method.
• amino acids amino acids
• HORSCH R.B Commercialization of genetically engineered crops. The production and uses of genetically transformed plants. Chapman 1 Hall LTD. London UK 19947, 99-103.
• ISHIDA ET AL. (1996), Nature Biotechnology, vol. 14: 745-750. • JEFFERSON, 1987, Plant Molecular Biology Reporter, vol.5: 387-405.

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