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
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis

Definitions

• the invention relates to means for regulating reproductive development in cultivated plants. More particularly, the subject of the invention is the development of plants that reproduce totally or partially by gametophytic apomixis, that is to say asexually via seeds.
• Gametophytic apomixis is a form of asexual reproduction by seed. It exists in many angiosperms, and nearly 400 apomictic species have been identified. However, apomictic plants are not found in the main cereals grown (maize, wheat, or rice), but only in wild plants, some forage species, and some fruit species. Apomixis is a genetically controlled mechanism. Apomictic plants develop female gametes without previous meiosis. The gametes thus formed contain a genome identical to that of the somatic tissues from which they are derived. The development of the embryo from these gametes is done without fertilization by a male gamete, ie by parthenogenesis. The genome of the embryo thus formed is therefore strictly identical to that of its parent plant, without paternal contribution. Apomixis is therefore a mode of cloning by seed that ensures the perpetuation of identical genotypes across generations.
• apomixis in a controlled manner in cultivated species offers many potential applications. These applications concern the propagation of unstable genotypes, the control of pollen contaminations, plant breeding methods, and commercial seed production methods.
• the work of the inventors in this field has shown that it is possible to induce an entirely or partially apomictic phenotype in maize by manipulating the expression of several genes that are collectively involved in the regulation of gene expression in organs. female reproductive (eggs) of corn. The seeds produced escape meiotic reduction and are fertile. These results apply advantageously to other crops such as rice or wheat.
• the invention therefore relates to the use of specific nucleotide sequences whose manipulation allows the development of plants reproducing totally or partially gametophytic apomixis. It is also intended to provide a method of producing apomictic plants.
• the invention aims to use apomixis in a controlled manner in cultivated species with sexual reproduction to develop many applications as will be explained below.
• the invention thus aims, for the production of partially or completely apomictic plants, the use of a gene coding for a DNA Methyl Transferase motif protein. It is more particularly a gene of the family of DNA Methyl Transferases encoding a protein sequence SEQ ID No. 1 or SEQ ID No. 5.
• the invention relates to the use of the gene DMT 103 from the family of DNA methyl transferases corresponding to the sequence SEQ ID No. 2 or the transcript of such a gene corresponding to the sequence SEQ ID No. 3 , or the ORF of sequence SEQ ID No. 4.
• the invention relates to the use of the DMT102 gene of the family of methyl transferase DNAs, corresponding to the sequence SEQ ID No. 6, or the transcript of such a gene corresponding to the sequence SEQ ID No. ° 7, or the ORF sequence SEQ ID No. 8. These genes are expressed specifically in the eggs where the reproductive cells are determined.
• the invention also relates to a method for inducing, in cultivated species such as maize, rice or wheat, an entirely or partially apomictic phenotype, characterized in that it comprises the targeted inactivation, by a transposable element, for example of Mutator type, a gene, a gene transcript or its ORF, as defined above, and the identification of the mutated locus.
• a transposable element for example of Mutator type, a gene, a gene transcript or its ORF, as defined above, and the identification of the mutated locus.
• apomixis in a controlled manner in cultivated species offers many potential applications. They concern the propagation of unstable genotypes, control of pollen contamination, methods of plant breeding, and methods of commercial seed production.
• the first application concerns the clonal propagation, by seed, of genetically unstable genotypes. This is the case in particular of all hybrid plants; these hybrid plants produce, by genetic mixing during the meiosis and the fertilization of the progenies which are different from each other, and different from their mother plant. This is still the case for cultivated species with unstable ploidy levels in meiosis, such as triploid forms.
• Apomixis also offers new perspectives in plant breeding. It would make it possible to use as a new variety any genotype selected as interesting, since it is a genetically determined criterion, whatever the genetic structure, since this one, since it is apomictic, becomes genetically stable. It is therefore possible to develop varieties directly from hybrid forms, possibly interspecific ones, by avoiding the stabilization steps currently required, such as the successive stages of self-fertilization, or the production of doubled haploids. This method therefore saves a considerable amount of time, but also certainly opens the door to the introduction of genetic materials that are totally new to selection programs, and in particular to genetic materials that, in sexual plants, induce high sterility. This is the case, for example, with most interspecific crosses. A very important application is the production of hybrid seeds.
• apomictic plants In the case of apomictic plants, however, one could consider producing the seeds directly from hybrids, thus with much higher yields, using 100% of the available area, without the need to control pollination, and without castration.
• the interest of using apomixis for seed production is very significant in species such as maize, where hybrid forms are already produced, for reasons of cost reduction, but also in self-pollinated species, such as for example, wheat or rice, where large-scale controlled hybridizations are difficult.
• the production of some apomictic hybrids would be sufficient to initiate large-scale production of hybrid, genetically stable seeds.
• Plants or seeds of partially or totally apomictic plants of cultivated species such as maize, rice and wheat, characterized in that they comprise inactivated alleles of a gene, as defined above, also fall within the scope of the invention.
• plants or seeds of plants of the invention are advantageously such as obtained not inactivation of the gene by mutagenesis or according to the method as defined above, to induce in the cultivated plants an entirely or partially apomictic phenotype.
• FIGS. 1 to 6 represent, respectively, FIG. 1: the comparative expression profiles of the chromatid regulators
• FIG. 2 the comparison of protein sequences with the alignment of the protein sequences of DMT 102 and CMT3, and DMT 103 and DRM2;
• FIG. 3 the expression profile of the two genes by mR A in situ
• FIG. 4 the structure of the two genes DMT 102 and DMT 103;
• FIG. 5 phenotypes of mutant plants with production of multiple embryonic sacs
• sequences SEQ ID Nos. 13 and 14 correspond to mutants of DMT 103-1042 and DMT 103-1342.
• the gametophyte is the haploid structure that develops from the products of meiosis, and contains mature gametes. It is the embryonic sac on the female side, and the pollen grain on the male side. In maize, the egg contains only one embryonic sac.
• Gametophytic apomixis refers to a form of seedless asexual reproduction in which gametes produced in female gametophytes have not undergone meiotic reduction, and therefore have the same ploidy and genetic makeup as the parent plant. Gametophytic apomixis involves two successive stages: apomeiosis and parthenogenesis.
• “Apomeiosis” refers to the mechanisms by which apomictic plants escape meiosis.
• Non-reduction refers to gamete formation in the absence of meiotic reduction.
• Non-reduced gametes are therefore gametes that develop in the absence of meiosis, or through non-reductional meiosis. Apomeiosis is therefore the specific form of non-reduction observed in apomictic plants.
• Disposy is a specific form of gametophytic apomixis, in which apomeiotic gametes develop from the same cells that participate in sexual reproductive development, that is, the archesore.
• Aposporia is a specific form of gametophytic apomixis, in which female gametophytes and apomeiotic gametes develop from the somatic cells of the ovum. In aposporic plants, sexuality and apomixis coexist functionally, and so we typically find several embryo sacs in the same egg, either from sexuality or aposporia.
• Parthenogenesis refers to the development of embryos without fertilization and without paternal genetic contribution.
• EXAMPLE 1 Regulatory Identification of the Deregulated Chromatin Structure in Apomictic Plants
• the experiment described hereinafter relates to the comparison of the expression profile of genes involved in the determinism of chromatin structure between sexual plants and apomictic plants.
• ARs of samples corresponding to apomictic and sexual eggs of the following stages of development were isolated; ova containing a megaspore mother cell, ovules containing a functional megaspore, and eggs at the time of fertilization.
• the sexual plants used are two reference maize lines, B73 and W23.
• Apomictic plants are hybrid forms obtained by crossing an apomictic plant of the species Tripsacum dactyloides, a wild relative of corn in which is found apomictic forms, with a sexed maize; these plants were then backcrossed several times over maize, selecting apomictic progenies, until plants with a diploid corn genome, and a haploid triptychic haploid genome were found.
• the analysis was done in two stages, first with the selection of genes expressing themselves specifically in the reproductive tissues; then, for these selected genes, the analysis of their expression profile at the various stages mentioned above for the apomictic and sexual forms.
• the alteration of the expression profiles may imply the total absence of expression in one form of reproduction relative to the other, this is the case, for example, of DMT 102 and CHR 106, the expression of which is completely abolished. in apomictic plants.
• EXAMPLE 2 Biological Function of Genes, Identified by Sequence Homology
• CHR 106 is a maize homolog of DDM1 in Arabidopsis, an enzyme involved in the maintenance of DNA methylation.
• DMT102, DMT103 and DMT107 are the homologs in corn of respectively CMT3, and DRM2 or DRM1 in Arabidopsis.
• DRM1 and 2 and CMT3 act partially redundantly in the control of asymmetric methylation (at the CHH or CHG site).
• MBD 109 is a methyl-binding domain (Methyl Bindind Domain) protein of unknown function, but is therefore likely to act on methylated sites.
• CHR120 is a maize homolog of MOM1 in Arabidopsis, a gene involved in silencing mechanisms.
• HXA102 is a corn homolog of AtADA2 in Arabidopsis, a component of the ADA histone acetyltransferase complex.
• SDG110 is a histone methyl transferase of unknown function.
• DMT102 and DMT103 correspond to an extremely well-characterized pathway in Arabidopsis, called the RdDM pathway (for RNA-dependent DNA methylation).
• DMT102 and DMT103 are respectively the homologues of CMT3 (CHROMOMETHYLASE 3) and DRM1 and 2 (DOMAIN REARRANGED METHYL TRANSFERASE 1 and 2) in Arabidopsis.
• the tissue expression profiles of these two genes (DMT102 and DMT103) were analyzed by in situ hybridization of RNA probes in a sexed corn.
• the results are illustrated in FIG. 3.
• the hybridization profiles obtained confirm the high specificity of expression detected by RT-PCR: the two genes express themselves very specifically during targeted stages of reproductive development. Thus, a signal is detectable for DMT102 immediately before and then during meiosis. DMT103 is detected only during gametogenesis.
• In situ data also show that, at the tissue level, these two genes are expressed only in a very limited number of cells, corresponding for each egg to the reproductive cell (the archesore, the mother cell of the megaspore and meiocytes during sporogenesis, the gametophyte during gametogenesis), and on the other hand to a small number of cells surrounding the reproductive cell.
• the mutant line corresponds to the insertion of a transposon of the Mutator family into the methyltransferase domain of the protein.
• the mutation results from the substitution of several amino acids in essential sites: R-49-I, R-272-Q, C-182-Y (residue in the wild-form - position - residue in the mutant).
• the mutant forms of DMT103 produce multiple embryonic sacs in a single egg.
• Figure 6 gives the phenotypes of mutant plants with production of unreduced gametes: A) the size of male gametophytes is in plants highly correlated at the level of ploidy gametes they contain. We can quickly assess the ability of a plant to produce unresolved gametes by microscopic observation of mature gametophytes, here the pollen grains.
• WT corresponds to a wild plant, the line W23.
• dmtl02-mu and dmtl03 are two mutant forms of DMT102 and DMT103, respectively, and clearly produce gametophytes of varying sizes, as illustrated by the frequency graph B).
• the frequency of these gametes in W23 wild plants, and the two mutant forms is quantified in C).
• the relationship between seed size and ploidy level is demonstrated by flow cytometric analysis (D).

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• 2009
  • 2009-11-09 FR FR0905375A patent/FR2952276A1/fr not_active Withdrawn
• 2010
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