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WO2011054998A2 - Modification de l'expression de la protéine della ou orthologue pour modifier le modèle de croissance des plantes et le contenu de métabolites du fruit - Google Patents

Modification de l'expression de la protéine della ou orthologue pour modifier le modèle de croissance des plantes et le contenu de métabolites du fruit Download PDF

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WO2011054998A2
WO2011054998A2 PCT/ES2010/070719 ES2010070719W WO2011054998A2 WO 2011054998 A2 WO2011054998 A2 WO 2011054998A2 ES 2010070719 W ES2010070719 W ES 2010070719W WO 2011054998 A2 WO2011054998 A2 WO 2011054998A2
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genetically modified
plant
sidella
della
protein
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PCT/ES2010/070719
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WO2011054998A3 (fr
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Antonio Granell Richart
José Luís RAMBLA NEBOT
Cristina MARTI IBAÑEZ
Abdel Bendahmane
Florence Piron
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Consejo Superior De Investigaciones Científicas (Csic)
Universidad Politécnica De Valencia (Upv)
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Publication of WO2011054998A3 publication Critical patent/WO2011054998A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention describes new functions of the SIDELLA gene of Solanum lycopersicum as well as its uses and mutant versions of the gene associated with new phenotypes that involve the modification of the growth habit and metabolism of the plants that carry these alterations resulting in an alteration in the meta bo lites content of tomato fruit.
  • the final architecture of a plant depends on the number, size and size of the constituent elements (leaves, internodes, flowers and fruits) and the way in which they are inserted into the general body of the plant. Even more important than the above is the growth habit.
  • the productivity of a plant is affected by genetic, physiological and environmental factors, but the architecture and growth habit are key determinants.
  • the tomato plant (Solanum lycopersicum) is from the Solanaceae family
  • the dwarf shrub varieties are a subgroup within the determined varieties characterized by their smaller size and for producing fruits of the "cherry” or "cherry” type.
  • the intensity of the flavor properties of tomato fruit is determined by the amount of sugar (mainly fructose and glucose), by the content of organic acids (mainly citric and malic) and the composition of volatile compounds.
  • sugar mainly fructose and glucose
  • organic acids mainly citric and malic
  • the difference in flavors between varieties is explained by the difference in the quantitative proportions of volatile substances, many of which develop during ripening.
  • Patent application ES 2235335 T3 describes a method of manipulating the aroma of tomato products, which consists of incubating tomato pieces with an enzyme with alcohol dehydrogenase activity and an alcohol dehydrogenase cofactor; as well as the products obtained by this procedure.
  • Sympodial growth plants have a peculiar lateral growth type in which the apical meristem ends (aborts or differs into an inflorescent flower / meristem) but the plant continues to grow from the axillary meristem normally located below the upper leaf, which is repeated after a few leaf nodes, causing continued apical growth.
  • Tomato plants exhibit a sympodial growth pattern characterized by alternating buds of vegetative and reproductive organs. This pattern is established after an initial period in which only vegetative organs are produced (stems / between nodes and leaves in the aerial part) which in the case of tomato involves the production of about 5 to 20 leaf nodes that ends the formation , in the apical meristem of the main outbreak, of an inflorescent meristem that supposes the termination of said main symposium. The growth of the plant does not stop there, but continues from the axillary bud located in the axilla of the leaf closest to that new inflorescence.
  • tomato plants that carry the recessive mutation self-pruning sp (sp / sp) in homozigosis only produce two or three sympodial shoots after the first inflorescence, each consisting of 2 or 3 leaves and an inflorescence, after which they produce 2 inflorescences in a row and growth ceases.
  • This mutation confers a "determined" growth habit that, in addition to affecting the size of the plant, results in fruit ripening and ripening more concentrated over time than occurs in indeterminate growth plants (Stevens and Rick, 1986; Yeager , 1927).
  • SP self-pruning locus
  • CEN Centroradialis
  • TTL1 Terminal Flower
  • SP belongs to a small gene family of six members (SP, SP21, SP3D, SP5G, SP6A, and SP9D) distributed in five chromosomes (Carme l-Goren et al., 2003), although the cause of the sp mutation that is on chromosome six is the only one that can control the passage between determined and indeterminate growth habit.
  • SP paralogs in tomato are not very characterized but they seem to affect the expression of the determined phenotype by modifying the moment at which the plant is determined (case of intrusions of SP9D and SP5G of pennelli in M82 (Carmel-Goren et al., 2003 ; Eshed and Zam ⁇ r, 1995; Jones et al., 2007)
  • the self-pruning mutation has been introduced in most of the industry tomato varieties, since it favors mechanical harvesting (Hanna et ai, 1966; Friedland and Barton, 1975; Stevens and Rick, 1986; Yeager, 1927) and there are no alternative methods or that modulate the expression of SP.
  • DELLA proteins have been described as key elements that would integrate both external and mediated signals by hormones, adapting plant growth to environmental needs and conditions. DELLA proteins are members of a GRAS subfamily of regulators that appear to act at the nuclear level as repressors of development and growth processes.
  • DELLA proteins a family of five members in Arabidopsis, but only one in rice
  • the blockade in the growth exerted by DELLA proteins would be eliminated by targeted degradation of the DELLA protein (Dill et al., 2001; Dill and Sun, 2001; Lee et al., 2002; Peng et al., 1997; Richards et al., 2001; Wen and Chang, 2002).
  • the mechanism of action underlying the effect of inducing the growth of phytohormones gibberellins would be based on the fact that after the union of GA to the GID receptor, a conformational change of the protein would take place that would favor its interaction with DELLA and increase its affinity to be degraded by the 26S proteasome. (Murase et al, 2007; Ueguchi-Tanaka et al, 2007; 2008).
  • DELLA route integrates the floral transition in response to light, ethylene and auxins among others and would act affecting the stability of DELLA proteins, their transcription or indirectly affecting the levels of GAs (Achard et al., 2006; Achard et al., 2003; Fu and Harberd, 2003; Oh et al., 2007).
  • the present invention provides genetically modified plants with the expression of DELLA proteins or altered orthologue thus producing an inhibition in the repression of the development and growth of plants in comparison to corresponding plants not genetically modified. Due to the alteration in the expression of DELLA, by silencing or mutation, genetically modified plants will see their sypodial growth pattern and / or the content of metabolites and / or volatile substances in the fruit altered.
  • the present invention also relates to the fruits and seeds of genetically modified plants as well as the method used to obtain said plants.
  • DELLA is regulated by the described SIDELLA gene and has been used by the authors of the present application (Marti ef al. 2007), however the regulation of its expression to alter the growth pattern and / or to alter the content of the metabolites of the fruits are new functions, as well as their applications.
  • An orthologous gene is known (similar to belonging to two species that have a common ancestor) of the SIDELLA gene, more specifically it is the GAI Arabidopsis tomato gene (Solanum lycopersicum).
  • the GAI gene has been cloned and mutated and the expression of such genes in plants affects their growth, specifically achieving dwarf plants that are useful for reducing the losses that They take place during harvest storage.
  • SIDELLA gene in tomatoes is important in determining the number of symposium repeats, and therefore the size and number of flowers / fruits on the main axis. This should affect the total productivity (number and size of fruits produced).
  • modifications in this gene affect the metabolic content of the fruits as exemplified by the content of volatile compounds associated with the aroma of the fruit.
  • a new function described here is relevant in plants whose main axis growth stops with the first flower to continue growing from the axillary bud (called a simpodial) below the new flower.
  • the new function has been revealed by the authors of the present application in studies with the SIDELLA gene.
  • the expected growth phenotype that is reflected in the length of internodes is observed.
  • the important thing about the present invention is that tomato plants that carry the sp mutation and therefore have a certain growth, that is to say, the growth of the main axis stops after a small number of 2 or 3 simpodial repetitions (metamer consisting for example in 2 leaves and one inflorescence) after the first flower, increase the number of sypodia or even become undetermined when the expression of SIDELLA in transgenic plants is blocked with different efficiency. This occurs without significantly modifying the flowering time and suggests an alternative way to sp to alter the growth habit of the tomato and by extension of other plants of similar growth (i.e. simpodial).
  • the architecture of the plant and the growth habit are modified by acting at the DELLA level (or at the level of orthologous proteins) ie by transgenesis by altering the level of SIDELLA (or orthologous gene) or by introducing mutations in the SIDELLA gene (or orthologous gene) that alter the growth pattern.
  • object of the present invention is the production and identification of mutant versions in that gene (or orthologous gene) that provide various intensities of the phenotype of interest.
  • the results of the present application have been obtained using tomato (Solanum lycopersicum) as an example, but they may be useful for other plants of specific or non-sypodial growth.
  • a method to associate the character with the mutations is also described, thus opening the possibility of identifying new mutants in the gene that give different versions of the phenotype that can adapt to what the producer needs.
  • silencing and mutations are of commercial and / or agronomic interest. It can be very important for those interested in altering the number of symposia buds in plants with that type of growth, including but not limited to solaneaceae, cucurbitaceae such as melon, woody, trees, and obviously ornamental as orchids, among other plants of interest. .
  • the first object of the invention relates to genetically modified plants with the altered expression of the DELLA protein or ortholog characterized in that the alteration in the expression of the DELLA protein or ortholog produces an inhibition in the repression of the development and growth of the plants in comparison to corresponding plants not genetically modified.
  • the alteration in the expression of DELLA of the object of the invention is obtained by silencing the SIDELLA or ortholog gene or by mutation of the SIDELLA or ortholog gene.
  • silencing of the SIDELLA or ortholog gene is obtained by employing a gene construct that is based on an antisense copy of the SIDELLA or ortholog gene controlled by the 2XCaMV35S promoter.
  • the mutation in the genetic sequence of SIDELLA is due to the addition and / or insertion and / or deletion and / or substitution of one or more nucleotides of the SIDELLA or orthologous gene, namely, the mutation consists of the nucleotide replacement guanine with adenine in nucleotide 458, called the TILLING 1 mutation (SEQ ID No.3) or the replacement of the guanine nucleotide with adenine in nucleotide 1498, called the TILLING 2 mutation (SEQ ID No.5) or the replacement of the cytosine nucleotide with thymine in nucleotide 994, called the TILLING 3 mutation (SEQ ID No.7) or the replacement of the cytosine nucleotide with thymine in nucleotide 661, called the TILLING 4 mutation (SEQ ID No.9).
  • the genetically modified plants of the present object of the invention exhibit a sypodi
  • genetically modified plants with the altered expression of the DELLA protein or ortholog of the present object of the invention are characterized in that the content of metabolites and / or volatile substances associated with the aroma of the fruits is increased and / or decreased with respect to to corresponding plants not genetically modified.
  • the increase in metabolite content refers in a particular embodiment to the increase in sucrose, tyrosine, asparagine, isoleucine, threonine, proline, pyroglutamic acid and myo-inositol of an order of 100-400%, and / or that of fructose and glucose in the order of 10-30%, and / or that of volatile substances associated with the aroma of the fruits ⁇ -2-hexenal, 1-pente-3-one and P.
  • the decrease in the content of volatile substances associated with the aroma of the fruits refers to a decrease in 3- methylbutanol, 1-nitro-2-phenylethane, 2-phenylethanol, phenylacetaldehyde and 2-isobutylthiazole in an order of 80-90%, and / or that of volatile substances not associated with the aroma of fruits geranylacetone, terpineol, linalool, benzyl alcohol, eugenol, benzylnitrile, 2-methyl-1-butanol and 2-methyl-1-propanol in an order of 50-100% with respect to corresponding plants not genetically modified.
  • the genetically modified plants of the present object of the invention are plants with a commercial and / or agricultural interest and are selected from the group of Solanaceae, Cucurbitaceae, Orchids, Woody, among other plants of interest.
  • the plants of the invention belong to the Solanaceae family, being specifically a Solanum lycopersicum tomato plant.
  • a second object of the invention relates to the fruits obtained from genetically modified plants according to the previous object of the invention with a content of certain metabolites and / or volatile substances associated with the increased aroma and / or the content of other meta balls and / or substances volatile associated with the diminished aroma with respect to the fruits of corresponding plants not genetically modified.
  • the increase in the metabolite content refers in a particular embodiment to the increase in sucrose, tyrosine, asparagine, isoleucine, threonine, proline, pyroglutamic acid and myo-inositol of an order of 100-400%, and / or that of fructose and glucose in the order of 10-30%, and / or that of the volatile substances associated with the aroma of the fruits ⁇ -2-hexenal, 1-penten-3- one and P.
  • the decrease in the content of volatile substances associated with the aroma of the fruits refers to a decrease in 3- methylbutanol, 1-nitro-2-phenylethane, 2-phenylethanol, phenylacetaldehyde and 2-isobutylthiazole in an order of 80-90%, and / or that of volatile substances not associated with the aroma of geranilacetone, terpineol, linalool, benzyl alcohol, eugenol, benzylnitrile, 2-methyl-1-butanol and 2-methyl-1-propanol in a order of! 50-100% with respect to corresponding plants not genetically modified.
  • Another object of the invention relates to the seeds of genetically modified plants according to the first object of the invention, in which the expression of the proteins D E L L A u o rtó I og a is found in Ite rad a.
  • Another object of the invention relates to the method of obtaining genetically modified plants with the altered expression of the DELLA or orthologous protein comprising the following steps: a) transform the genome of a plant cell, regenerate the plant from the cell of stage a), obtain seeds of the transformed plant containing the modified genome and, grow at least one of the seeds of stage c ) to obtain one that contains the altered expression of the DELLA protein or ortholog.
  • step a) of the present object of the invention consists of a directed mutagenesis whose realization is obtained by silencing the SIDELLA gene using the asDELLA gene construct that is based on an antisense copy of the SIDELLA gene controlled by the 2XCaMV35S promoter.
  • step a) of the present object of the invention consists of a directed mutagenesis whose embodiment consists in modifying the native sequence of the SIDELLA gene to the sequence of the mutated SIDELLA gene selected from the group of mutations called TILLING 1 (SEQ ID No 3 ), TILLING 2 (SEQ ID No 5), TILLING 3 (SEQ ID No 7) and / or TILLING 4 (SEQ ID No 9).
  • step b) is optional and in another embodiment said transformation is mediated by Agrobacterium.
  • step a) consists of an non-directed mutagenesis comprising the following stages: i. expose the plant to a mutagenic agent, preferably the tilmetanesulfonato extract the DNA from each of the families and combine following a 3D strategy, track the SIDELLA gene in each family by nested PCR and universal primers,
  • step (iv) of the present embodiment 4 allelic forms of SIDELLA were selected, namely those named: TILLING 1 (SEQ ID No 3), TILLING 2 (SEQ ID No 5), TILLING 3 (SEQ ID No 7) , TILLING 4 (SEQ ID No 9) ..
  • Figure 1 Structure of the gene construct introduced in tomato to decrease the levels of SIDELLA in transgenic plants.
  • CaMV poly (A) + CaMV polyadenylation sequence.
  • Nptll gene that confers resistance to kanamycin, with the promoter and terminator of nopaiin synthetase (pnos and tnos).
  • FIG. 2 Effect of the strategy on SIDELLA levels in asDELLA transgenic plants. Analysis of the expression levels of asSIDELLA and SIDELLA in internodes of transgenic and wild plants by semi-Peruvian RT-PCR. The figure confirms the expression of asSIDELLA in the transgenic lines (lines 5A, 24B and 7G). At the same time the decrease of the endogenous levels of SIDELLA in these same lines is checked in comparison with a wild plant (wt). As a load control, the RT-PCR reaction for actin was used.
  • Figure 3 Effect of the strategy on the growth and production of sympodial outbreaks in asDELLA transgenic plants.
  • A Effect on the total height of the plants measured after 90 days.
  • B Effect on growth, number of internodes and their size over 120 days.
  • Figures 4-7 (A) Nucleotide sequences and (B) primary structure of the proteins corresponding to the mutated forms of SIDELLA identified by TILLING: Mutante TILLING 1 (figure 4), Mu ⁇ a ni e TILLING 2 (figure 5), Muiá ⁇ e TILLING 3 (figure 6) and Mu ⁇ a ni e TUILLING 4 (figure 7), where the muted nucleolides and amino acids are marked in boldness and underlined by the sequence of culinary SIDELLA determined M82.
  • FIG 4 In the TILLING 1 mutant (A) at the nucleotide level there is a substitution of guanine for adenine (at nucleotide 458) and (B) at the amino acid level there is a substitution of alanine for threonine (at amino acid 153).
  • Figure 5 In the TILLING 2 mutant (A) at the nucleotide level there is a substitution of guanine for adenine (in nucleotide 1498) and (B) at the amino acid level there is a substitution of glutamine for lysine (at amino acid 500).
  • FIG 6 In the MILLING TILLING 3 (A) at the nucleotide level there is a substitution of cytosine for you mine (at nucleotide 994) and (B) at the amino acid level there is a substitution of leucine for phenylalanine (at amino acid 332).
  • Figure 7 In the MILLING TILLING 4 (A) at the nucleotide level there is a substitution of cytosine for imimine (in nucleotide 661) and (B) at the amino acid level there is a substitution of leucine for phenylalanine (at amino acid 221).
  • FIGS 8-11 Stacking of the different TILLING mutants characterized and location of the mutation in the primary structure of the protein compared to other DELLA proteins of other plants to see the conservation degree of the affected amino acid. Except in the TILLING 1 mutant, all mutations occur in highly conserved regions of the protein.
  • Figure 12 M 82 wild plant phenotype compared to TILLING plants with point mutation in homozigosis or he ⁇ erozigosis in SIDELLA. An increase in the height of the mutant plants is observed, which present greater biomass and greater number of fruits.
  • the term "sypodial index” is the number of nodes (leaves) on the main bud until the inflorescence.
  • the "growth habit” (plant ha bit), also called growth pattern, according to the present invention is that presented by plants due to the composition, branching pattern, development and texture as well as the arrangement in space of the modular units consisting of leaves, internodes and flowers (fruits).
  • the "simpodial growth” is that presented by plants such as tomatoes, cantaloupe, many cucurbitaceae, legumes and orchids, etc., and which is characterized in that the growth of the main axis of the plant stops with the formation of the first inflorescence, to continue growing by differentiation and elongation from the differentiation of the bud corresponding to the bud located on the leaf just below that inflorescence. That axillary bud produces a number of leaves and a new terminal flower, to resume growth from the axillary bud below that new inflorescence and so on.
  • sypodial growth plants are the Solanaceae, Cucurbitaceae, Orchids, Woody, among other interest groups.
  • Determined growth is the growth habit characterized by the production of a limited number of sypodial shoots on the main axis, after the first flower, resulting in a plant of smaller height.
  • it is achieved by mutation in the self-pruning gene in homozigosis.
  • “semi-determined growth” is the habit that plants with certain growth have, in which they produce a larger number of symposia buds than the reference genotype under certain conditions.
  • the "indeterminate growth” is the one presented by many species (all wild ones related to tomato and many cultivated) of tomato and typical of all the "vineyards", it is characterized by a simpodial growth, which is repeated indefinitely on the main axis producing new shoots with each new terminal inflorescence.
  • transgenesis in the present invention, is the process by which a DNA sequence containing at least one region not present in the genome of the initial organism is introduced.
  • Genetically modified plant refers, in the present invention, to plants whose genetic material has been deliberately modified in order to modify the expression of the DELLA protein or ortholog.
  • the genetic modification can be carried out both by means of a directed mutagenesis and through non-directed mutations.
  • directed mutagenesis refers to a specific genetic modification, in which a nucleotide chain in the genome of a plant cell is specifically modified.
  • Non-directed mutagenesis refers to the mutagenesis of the cellular genome without directing the mutation, that is, it is not known in advance what mutation is going to be generated, where or the effect that said mutation will have on the cell and / or plant.
  • corresponding non-genetically modified plant (wild plant) refers in the present invention to plants whose genetic material has not been modified.
  • Orthologous gene in the present invention refers to homologous genes in different species that encode “orthologous proteins” that catalyze the same reaction (ie, with the same function) in plants.
  • new functions and uses of the SIDELLA gene of So ⁇ anum lycopersicum are described together with mutant versions of the gene associated with the new phenotypes. These new genotypic characters include the modification of growth habit and metabolism. Genetically modified plants in SIDELLA or allelic mutant forms of this protein can be used to increase the number of sypodial repeats in a given genetic background with the corresponding increase in the number of inflorescences and fruits, and on the other hand produce fruits with a metabolic content. Modified ico, including that of the volatiles associated with the aroma.
  • the present invention is related to the genetic control of growth and more specifically to the growth habit that determines the determined or non-determined growth of a plant. More specifically, this invention is related to the use of the SIDELLA gene to modify that aspect of development through genetic engineering and TILLING strategies followed by marker-assisted improvement using the mutant forms of this gene.
  • a first aspect of this invention is based on the identification of a new function of the DELLA protein not previously described and which provides a way to alter the determined growth habit of plants (in our case, tomato plants carrying the mutation sp / sp).
  • This way of altering the growth habit in tomato is to use an asDELLA construction to decrease levels endogenous to the SIDELLA regulator in transgenic plants.
  • the transgenic plants thus produced have a semi-determined to indeterminate plant phenotype with a greater production of flowers and fruits on the main axis, without significantly altering the flowering time.
  • Another aspect of the present invention and since DELLA proteins degrade after treatment with the gibberellin hormone GAs (Dill et al. 2001; 2004; Gubler et al.
  • the new SIDELLA function can be conferred by transgenesis analogously to that resulting from the use of an asDELLA strategy as described in example 1 or by over-expression of mutated versions, including those of function gain (gai type suppression in asDELLA or related).
  • a method is proposed to identify mutants, derivatives, variants and alleles of that protein from mutagenized plants and that result in new functional characteristics that modify in different degree the number and composition of the simpodial repeats, thus producing plants. with a number of flowers and size adapted to the needs.
  • the changes in the protein may be one or more of the following: addition, insertion, deletion or substitution of one or more nucleotides that results in changes in the amino acid sequence or not.
  • the present invention also provides the nucleoacidic construct or vector comprising a promoter from which the SIDELLA sequence is expressed in antisense orientation.
  • the construction or vector is designed for expression in a cell, especially plant type. Said plant cell expressing that antisense construct is also included in this invention.
  • This construction when inserted into the genome of the plant directs the expression of the complementary mRNA chain of SIDELLA causing the reduction of the levels of the endogenously expressed coding chain. This decrease can also be obtained by other methods such as RNAi, micro R NA, etc.
  • Said construction or vector containing the SIDELLA in antisense, RNAi, etc. will generally contain a promoter or other regulatory sequence.
  • the present invention can be carried out in any plant, especially of simpodial growth, apart from tomato (ie cucurbitaceae or orchids, etc.) in which the SIDELLA ortholog can be isolated and identified by PCR with preserved oligos and carrying out a similar approach.
  • nucleic acid libraries can be tracked with heterologous probes. People skilled in the art can carry out these procedures without problems and carry out genetic constructions and transformation in a similar way to obtain an alteration of the phenotype described in those plants.
  • mutant sequences of SIDELLA are presented that confer alterations in the new function described and that result in tomato plants with a greater number of symposia than the variety provided by the genetic background and with a greater number of inflorescences and alteration of the symposium composition.
  • DELLA or orthologous protein
  • DELLA or orthologous protein
  • SIDELLA or orthologous gene
  • the organoleptic, nutritional and health characteristics of the fruits are a direct consequence of the content in a series of molecules present in it.
  • the flavor and aroma of tomato is related to the concentration and relative amounts of sugars (mainly glucose and fructose) and organic acids (malic, citric) and volatile compounds that contribute to the aroma (at least 14 volatile compounds).
  • sugars mainly glucose and fructose
  • organic acids malic, citric
  • volatile compounds that contribute to the aroma (at least 14 volatile compounds).
  • the flavor and aroma are characters often forgotten in the most recent improvement programs, where the emphasis has been on productivity, long life of the fruits, etc., with the consequent social demand for fruits with better or different flavors.
  • the present patent it is shown that it is possible to modify the relative content of volatile compounds, sugars and amino acids in tomato fruit acting at the level of SIDELLA.
  • the method consists of using natural or generated variability through mutagens and identifying mutated variants by TILLING or similar.
  • TILLING Targeting Induced Local Lesions In Genomes
  • This technology provides a way to obtain mutants in a gene and in our case it was used to find mutants in SIDELLA, since we knew that TILLING combines mutagenesis protocols with POR and a method for detecting DNA polymorphisms.
  • any cell, especially plant that contains the construction directed to modify the levels of expression of DELLA or that contains specific mutants in DELLA affected in this new function.
  • the present invention also comprises the plant comprising said carrier cell of the construction or mutation in SIDELLA.
  • the present invention provides any clone of said plant, self-fertilizing or hybrid seed and its descendants and any part thereof as cuttings, seeds, etc. that can be used to propagate such material.
  • the present invention also provides a method for influencing said new phenotype, such as treatment with GAs or activators or inhibitors of its synthesis or mechanism of action.
  • the present invention also provides a method for using function gain mutations in DELLA (deletions in the gal mutant type protein) to alter the phenotype in the opposite direction (decrease the number of sypodia or flowers by simpodial repetition).
  • a method is provided to increase the number of symposia by maintaining (specific mutants or higher levels of repression) or not (high level of repression or null mutants) the determined character and the number of flowers per symposium, which affects in the number of fruits.
  • the decrease in DELLA levels or the expression of mutated forms of DELLA were made induciblely or by specific promoters.
  • constructions such as those indicated above or mutated versions such as those described controlled by a specific or inducible promoter. This would affect the number of symposia and the composition of the fruits in a more directed way
  • Example 1 Increase in the number of simpodial repetitions by inhibiting the expression levels of the SIDELLA gene in transgenic tomato plants.
  • the coding sequence of the SIDELLA gene (SEQ ID No. 1 and 2) was placed in antisense orientation under the control of the constitutive 35S promoter of the cauliflower mosaic virus (CaMV) (see scheme Figure 1) (The SIDELLA cDNA sequence It has already been published and used in this construction), and introduced into tomato plants (Solanum lycopersicum UC82 sp / sp) by Agrobacferium-mediated transformation following established procedures (Ellul et al. 2003).
  • the UC82 (sp / sp) plants that carry the self-pruning mutation in homozigosis have the characteristic determined habit, forming the first inflorescence after about 10 leaves and finishing their growth after producing 3 metamers. sypodial,
  • the transgenic lines of tomato UC82 (sp / sp) carrying the asDELLA construction show decreased levels in SIDELLA ( Figure 2) and an altered bearing (growth habit) ( Figure 3, tables 1 and 2).
  • TABLE 1 Effect of the decrease in the expression of SIDELLA in transgenic plants on their height, number and length of internodes, flowering time and composition of metamers.
  • Table 1 shows the highest height of transgenic plants in relation to wild plants, as well as an increase in the number and length of their internodes. On the other hand it is verified that the flowering time, measured as number of leaves until the appearance of the first inflorescence, is only delayed somewhat in the 5A plants. As for the composition of the metameres, it is observed that in wild plants the inflorescences appear in consecutive leaves and the growth of the plants stops after three inflorescences, while in the asDELLA the inflorescence appears every 2 or 3 leaves and continues to form additional metamers indefinitely when the wild ones have stopped growing.
  • the cDNA corresponding to SIDELLA was isolated from a lambda ZAP expression library of tomato ovaries (Solanum lycopers ⁇ cum L. ve Rutge ⁇ emasculated one day before the anthesis.
  • the SIDELLA gene was screened from the colonies obtained (40,000) from the expression library using as a probe the complete coding region (cDNA) (1750 bp) of the gaidel mutated gene of Arabidopsis thal ⁇ ana (Atgaldet).
  • nitrocellulose filters obtained from the Lysis plates were performed at 46 ° C for 6 h.
  • the nitrocellulose filters were washed twice with 2 x SSC, 0.1% SDS at room temperature for 5 min and once with 0.1 x SSC, 0.1% SDS at 46 ° C for 22 minutes, 15 positive clones corresponding to the same gene were obtained, isolating and sequencing the longest of them (2389 bp.)
  • This clone contained an ORF of 1764 bp capable of synthesizing a protein of 588 amino acids with a molecular weight of 64, 45 KDa and one pu theoretical isoelectric number of 5.07.
  • the comparison of the deduced protein sequence with those existing in the databases showed that the isolated clone was a tomato ortholog of the DELLA genes
  • plasmid pBINJIT60 under the control of the 35S promoter of the tobacco mosaic virus (35S :: SIDELLAas).
  • the binary plasmid, pBINJIT60 was used to obtain transgenic Lycorpersicon esculentum plants by transformation with Agrobacterium tumefaciens.
  • This plasmid is constructed from the "cassette” of plasmid pJIT60, which provides the 35S promoter of cauliflower mosaic virus (CaMV) with the duplicated “enhancer” (2x35S), the multiple cloning site of pUC9 and the sequence of polyadenylation of the CaMV, [CaMV poly (A) +] (Guerineau et al., 1992), extracted as a Kpnl and Xhol fragment, and subcloned into the Kpnl and I left sites of the binary plasmid pBIN19.
  • CaMV cauliflower mosaic virus
  • 2x35S duplicated "enhancer”
  • PBIN19 contains the NPTII gene, which confers resistance to kanamycin, fused to the promoter and the nopaline synthetase terminator (pnos and tnos), included in the T-DNA (Bevan, 1984).
  • the resulting vector pBINJIT is approximately 13.2 kb in size and presents as unique cloning sites Sal ⁇ , BamHI and Smal.
  • To clone asDELLA antisense in this vector was amplified by PCR using the complete genome sequence of TGxC7 oligonucleotides (5 '-CCAGCACTTGTCATTCTTACC-3' SEQ. ID. No. 13) and TGxCS (5-CATCTCTCTCATTGTCTCTTCC-3 'SEQ. ID No.
  • the 1800pb product was digested with EcoRV and subcloned into the pBSK vector, choosing those clones in which SIDELLA had been cloned in antisense to subclone it into the binary vector pBINJIT60 using the Sma I / Sal I sites in order to reduce the levels Endogenous to SIDELLA, this construction was introduced in the strain LBA4404 of Agrobacter ⁇ um tumefaciens by electroporation for later use in the transformation of tomato from cotyledon explants (Ellul et al., 2003).
  • Example 2 Obtaining and identifying tomato SIDELLA alleles in M82 (sp / sp).
  • TILLING Targeting Induced Local Lesions In Genomes
  • This technology provides a way to obtain mutants in a gene and in our case it was used to find mutants in SIDELLA since we knew that TILLING combines mutagenesis protocols with PCR and a method for detecting DNA polymorphisms.
  • the TILLING method combines the induction of a large number of point mutations by chance caused by the Ethyl Methane Sulfonate (EMS) that produced a population of 12,000 2 families in the tomato variety determined M82 (Menda et al., 2004).
  • EMS Ethyl Methane Sulfonate
  • the DA was extracted from each of the families and combined following a 3D strategy to decrease the number of PCR reactions to be performed.
  • the SIDELLA locus of interest was amplified from each of the pools by means of a nested PCR and universal primers.
  • the first PCR amplification is a standard PCR reaction that uses specific primers of the SIDELLA target gene as indicated in (Qiu et al., 2004).
  • specific primers of the SIDELLA target gene as indicated in (Qiu et al., 2004).
  • SIDELLA-ext-F1 5'cattctctaatggtgctgttttcttc3 '(SEQ. ID. No. 15);
  • SIDELLA-ext-R1 5'aggtagctataagtggccgtgtatg3 '(SEQ. ID. No. 16);
  • SIDELLA-F1 5'gaaaagtaagatttgggaagaaga3 '(SEQ. ID. No.
  • SIDELLA-R1 5'ctaaaagcatggaagcttgtttgaa3 '(SEQ. ID. No. 18).
  • the amplification conditions were 1 min at 94 ° C and 30 cycles of (1 Os at 94 ° C, 20 s at 63 ° C, 1 min at 72 ° C) and 5 min at 72 ° C when SIDELLA oligonucleotides were used -ext-R1 and SIDELLA-ext-F1.
  • a microliter of the first PCR was used as a template for a second nested PCR amplification reaction, using a mixture of specific internal oligonucleotides bearing a universal M13 tail, combining with the universal M13 d primers.
  • M13F70Q CACGACGTTGTAAAACGAC; SEQ. ID. No. 19
  • M13R800 GG ATAAC AATTTC AC AC AGG; SEQ. ID. No. 20
  • IRD700 and IRD800 LI-COR®, Lincoln , Kansas, USA
  • This PCR reaction was carried out using each primer at the concentration of 0.1 ⁇ , and the following two-step cycle program: 94 ° C for 2 min, 10 cycles at 94 ° C for 15 s, a banding temperature of specific primers for 30 S and 72 ° C for 1 min, followed by 25 cycles at 94 ° C for 15 s, 50 ° C for 30 s and 72 ° C for 1 min, and finally an extension of 5 min at 72 ° C .
  • the detection of mutations in unpurified PCR products was carried out as indicated in Triques et al., 2007, except that the enzyme extract was used at a dilution of 1 to 10 000 and 0.6 ⁇ of digestion products were loaded with ENDOI in the sequencing gel.
  • the mutants were evidenced by occupying a different position in the sequencing gel, and by deconvolution of the different lines mixed in the pool.
  • the mutant present in the pool was confirmed individually and the mutation was sequenced following standard procedures.
  • TILLING 1 mutants SEQ. ID. No. 3 and 4
  • TILLING 2 SEQ. ID. No. 5 and 6
  • TILLING 3 SEQ. ID. No. 7 and 8
  • TILLING 4 SEQ. ID. No. 9 and 10
  • TILLING 1 SEQ. ID. No. 3 and 4
  • TILLING 2 SEQ. ID. No. 5 and 6
  • TILLING 3 SEQ. ID. No. 7 and 8
  • TILLING 4 SEQ. ID. No. 9 and 10.
  • TABLE 3 Summary of changes caused by TILLING mutations at the nucleotide and amino acid level, environment of amino acid change, type of mutation and effect on the growth habit of the mutation carrier plant.
  • the effect of these point mutations in SIDELLA is the change in the growth habit of the plant, going from being of a determined to semi-determined type, being nt: nucleotide; aa: amino acid; wt: wild; mt: mutation.
  • Example 4 Alteration in the content of metabolites in the fruit as a result of the modification in SIDELLA.
  • the metabolites analyzed have a determining effect on the organoleptic and nutritive qualities of tomatoes, since volatile compounds are responsible for the aroma, while some of the primary metabolites analyzed, including glucose and fructose sugars, organic acids Citric, malic and succinic, and several amino acids, have a decisive contribution on the flavor and nutritional content of the fruits.
  • the fiber used has a 65 ⁇ coating of polydimethylsiloxane-divinylbenzene (PDMS / DVB) (SUPELCO).
  • PDMS / DVB polydimethylsiloxane-divinylbenzene
  • SUPELCO polydimethylsiloxane-divinylbenzene
  • the vials were tempered at 50 ° C for 10 minutes and, subsequently, the fiber was exposed to the head space for 20 minutes at the same temperature.
  • the acquisition of volatiles in the fiber and subsequent desorption was performed automatically with a CombiPAL (CTG Analytics).
  • the detection was carried out using an Agilent 5975 B mass spectrometer in the Sean mode, in the range of m / z 35-220, at 7 scans / s, ionization source temperature 230 ° C and ionization energy 70eV.
  • the chromatograms were processed with the MSD ChemStation Enhanced Data Analysis software (Agilent Technologies).
  • the primary metabolite analysis was basically performed using the protocol described in Roessner-Tunali et al, 2003, which is detailed below, using the same plant material described above for the analysis of volatile elements.
  • the metabolites were extracted first. Approximately 250 mg of plant material was weighed, 3 ml of methanol and 120 ⁇ of a solution of the internal ribitol standard (0.2 mg / ml in water) were added, and vortexed vigorously for 20 s. It was transferred to a 5 ml glass vial, closed and incubated in a 70 ° C water bath for 15 min. Subsequently, 1.5 ml of water was added, vortexed, and centrifuged at 4000 rpm for 15 min. 50 ⁇ of the supernatant was taken, taken to a 1.5 ml polypropylene tube and dried in a speed-vac for 12 hours, at room temperature.
  • the internal ribitol standard 0.2 mg / ml in water
  • the extracted metabolites were derivatized as follows. 60 ⁇ of a solution of O-methylhydroxylamine hydrochloride (30 mg / ml in pyridine) was added, and kept at 37 ° C with stirring for 2 hours. After a centrifugation pulse, 120 ⁇ of N-methyl-N- (trimethylsilyl) trifluoroacetamide (STFA) and 12 ⁇ of a mixture of several fatty acid methyl esters (800 ng / ml each in chloroform), and kept at 37 ° C, under stirring, for 30 min. Finally, it was transferred to a 2 ml GC vial with 200 ⁇ insert, from which the injection was made.
  • STFA N-methyl-N- (trimethylsilyl) trifluoroacetamide
  • Arabidopsis F-box protein SLEEPY1 targets gibbere ⁇ lin signaling repressors for gibberellin-induced degradation. Plant Cell 16: 1392-1405.
  • the Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses. Genes Dev. 1 1: 3194-3205.

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Abstract

L'invention concerne la modification de l'expression de la protéine Della ou orthologue pour modifier le modèle de croissance des plantes et la teneur en métabolites du fruit. La présente invention concerne de nouvelles fonctions du gène SIDELLA de Solanum Lycopersicum ainsi que ses utilisations et des versions mutantes du gène associées à de nouveaux phénotypes qui supposent la modification du type de croissance et du métabolisme des plantes porteuses de ces modifications comme une certaine influence dans la teneur en métabolites du fruit de la tomate.
PCT/ES2010/070719 2009-11-07 2010-11-05 Modification de l'expression de la protéine della ou orthologue pour modifier le modèle de croissance des plantes et le contenu de métabolites du fruit WO2011054998A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102277360A (zh) * 2011-08-05 2011-12-14 中国农业大学 一种对樱della蛋白及其编码基因与应用
CN104558132A (zh) * 2015-01-14 2015-04-29 山东省农业科学院生物技术研究中心 花生della基因家族及其编码基因与应用
JP2015089368A (ja) * 2013-11-07 2015-05-11 国立大学法人 筑波大学 変異型植物
WO2019053725A1 (fr) 2017-09-18 2019-03-21 Futuragene Israel Ltd. Contrôle d'expression spécifique au tissu de polypeptides della

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102277360A (zh) * 2011-08-05 2011-12-14 中国农业大学 一种对樱della蛋白及其编码基因与应用
CN102277360B (zh) * 2011-08-05 2012-10-17 中国农业大学 一种对樱della蛋白及其编码基因与应用
JP2015089368A (ja) * 2013-11-07 2015-05-11 国立大学法人 筑波大学 変異型植物
EP3081641A4 (fr) * 2013-11-07 2017-05-10 University of Tsukuba Plante mutante
US10385357B2 (en) 2013-11-07 2019-08-20 University Of Tsukuba Mutant plant
CN104558132A (zh) * 2015-01-14 2015-04-29 山东省农业科学院生物技术研究中心 花生della基因家族及其编码基因与应用
CN104558132B (zh) * 2015-01-14 2018-06-19 山东省农业科学院生物技术研究中心 花生della基因家族及其编码基因与应用
WO2019053725A1 (fr) 2017-09-18 2019-03-21 Futuragene Israel Ltd. Contrôle d'expression spécifique au tissu de polypeptides della
US11555195B2 (en) 2017-09-18 2023-01-17 Futuragene Israel Ltd. Tissue-specific expression control of DELLA polypeptides
IL272713B1 (en) * 2017-09-18 2024-06-01 Futuragene Israel Ltd Tissue-specific expression control of della polypeptides
IL272713B2 (en) * 2017-09-18 2024-10-01 Futuragene Israel Ltd Tissue-specific expression control of della polypeptides

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