EP1802768A2 - Plante a teneur en lignine reduite par modulation de l'expression genique de dahps - Google Patents

Plante a teneur en lignine reduite par modulation de l'expression genique de dahps

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EP1802768A2
EP1802768A2 EP05789584A EP05789584A EP1802768A2 EP 1802768 A2 EP1802768 A2 EP 1802768A2 EP 05789584 A EP05789584 A EP 05789584A EP 05789584 A EP05789584 A EP 05789584A EP 1802768 A2 EP1802768 A2 EP 1802768A2
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giy
leu
ala
arg
plant
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Anders Jensen Boegh
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DLF Trifolium AS
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DLF Trifolium AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • 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
    • C12N15/8255—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 lignin biosynthesis

Definitions

  • TITLE A plant with reduced lignin by modulating DAHPS gene expression.
  • the invention relates to a plant or plant cell that comprises a stable modification of the genome, wherein the genome modification causes a stable reduction in the amount of mRNA level of a DAHP synthase that is expressed in relatively high amounts in stem tissue of the plant.
  • the plant may have reduced amount of lignin.
  • Lignins are complex phenolic polymers present in all vascular plants. They provide rigidity to conducting xylem elements and fiber cells. Lignins are composed Of C 6 C 3 units, principally p- hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, and are present in various proportions according to botanical, physiological, andcytological criteria. Throughout the plant kingdom, grass lignins appear to be particularly specialized because they contain not only H, G, and S units, but also additionalp-hydroxycinnamic units such as/7-coumaric and ferulic acids.
  • H principally p- hydroxyphenyl
  • G guaiacyl
  • S syringyl
  • Ferulic acid may be ester linked to wall polysaccharides and/or ether linked to G units, thereby forming bridges between lignins and polysaccharides, whereas />-coumaric acid is primarily ester linked to S lignin units in lignified walls.
  • transgenic grass plants wherein the transgenic plant (by use of anti-sense (AS) technology) expressed lower amount of enzymes that catalyzes the last steps in the biosynthesis of lignin.
  • AS anti-sense
  • enzymes are caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD).
  • COMP caffeic acid O-methyltransferase
  • CAD cinnamyl alcohol dehydrogenase
  • figure 1 herein For instance (Chen L. et al, Plant Biotechnology Journal (2003) 1, pp. 437 ⁇ 449) describes a transgenic forage grass (tall fescue) with reduced CAD activity and with decreased lignin content.
  • the transgenic grass has a 7.2 to 9.5 increased dry matter digestibility.
  • the article (Piquemal J. et al, Plant Physiol, December 2002, Vol. 130, pp. 1675-1685) describes transgenic maize with reduced COMT activity and with decreased lignin content.
  • lignification in dicotyledons has been more extensively studied and most of the known lignin biosynthetic genes have been employed in genetic engineering experiments. For review, see (Grima-Pettenati J, Goffner D (1999) Lignin genetic engineering revisited. Plant Sci 145: 51-65). Although lignification in grass species is likely to share a high degree of similarity to other angiosperms, the aforementioned structural specificity of grass cell walls may also involve a certain degree of grass-specific regulatory mechanisms. Different studied dicot plant species include tobacco, tomato, potato and Arabidopsis thaliana.
  • the enzyme 3-deoxy-7-phosphoheptulonate (DAHP) synthase (EC 2.5.1.54) (Formerly EC 4.1.2.15) is the first enzyme of the Shikimate pathway leading to biosynthesis of the aromatic amino acids.
  • the Shikimate pathway relates to the ability to synthesize aromatic amino acids de novo in bacteria, fungi, and plants (see figure 1 herein).
  • the aromatic amino acids Phenylalanine (Phe), Tyrosine (Tyr) and Tryptophan (Trp) are not only building blocks for protein synthesis but are also precursors for phenylpropanoid and indole derivatives.
  • These secondary compounds include hormones like indole acetic acid, pigments like anthocyanins, antimicrobial compounds like phytoalexins, and lignin, which play a vital role in plant defense, wound healing and maintaince of structural integrity and water transport capacity.
  • the problem to be solved by the present invention is to provide a transgenic plant that may have reduced lignin production.
  • the solution is based on that the present inventors have analysed plants in details and found that different tissues of plants expressed different amounts of specific DAHP synthases. Further, it was identified that some specific DAHP synthases were expressed in significantly higher amounts in stem tissue, when the plant were grown under normal natural conditions (e.g. without specific physical wounding). See working examples herein.
  • Plant stem tissue is known to be a tissue with particular high amount of lignin. Identification of specific DAHP synthases that are expressed in significantly higher amounts in stem tissue may be done in a number of ways. A suitable way is to first identify different DAHP synthase DNA sequences of a plant of interest and then measure expressed mRNA levels in different tissues (e.g. root and stem tissues).
  • DAHP synthases that are expressed in significantly higher amounts in stem tissue. Determination of mRNA levels may e.g. be performed by Northern Blotting or quantitative PCR. See e.g. working examples herein for an illustrative example in the forage grass tall fescue.
  • transgenic plant may have reduced amount of lignin by making a transgenic plant, wherein the amount of a DAHP synthase, expressed in high amounts in stem tissue, is reduced.
  • This may e.g. be done by anti-sense technology, wherein a DNA construct expressing a specific gene for the DAHP gene in anti- sense orientation is introduced into the plant.
  • specific deletions in the relevant DAHP gene may be made to obtain a plant that does not express the relevant DAHP gene. The specific way of doing it is not essential and it may also be done in other ways as further discussed herein.
  • the solution of the present invention which relates to down regulation of specific DAHP synthases that are relatively highly expressed in stem tissue, is significantly different from the prior art solutions.
  • the prior art strategies have focussed on down regulating enzymes that catalyzes the last steps in the biosynthesis of lignin, such as COMT and CAD.
  • the present invention is based on a strategy that is focussed on decreasing the quantity of basis material (aromatic amino acids) for lignin production in specific relevant tissue (in particular stem and leaf tissue).
  • basis material aromatic amino acids
  • a further advantage of decreasing the amount of specific DAHP synthases is that the amount of digestible sugar could subsequently increase in the plant. See figure 1 for an illustration.
  • a first aspect of the invention relates to a method for identifying a 3-deoxy-7- phosphoheptulonate (DAHP) synthase that is expressed in relatively high amounts in stem tissue of a plant comprising the following steps: (i): identifying DNA encoding sequences of two or more different DAHP synthase genes of the plant;
  • a DAHP synthase gene that in stem tissue is higher expressed compared to at least one other DAHP synthase gene of the same plant.
  • a DAHP synthase gene that is the relatively highest expressed in stem tissue may be identified.
  • DNA encoding sequences of step (i) may be fragments of the DAHP synthase genes. As understood by the skilled person one needs not to have the complete gene to perform the method. Fragments would be enough if the fragments are sufficient long to e.g. form basis for making adequate probes or primers to measure the mRNA level as described in the method.
  • the term "elongation growth stage of the plant” relates to a growth phase of the plant where the plant elongates and makes a relatively high amount of lignin in order to make the relevant structures and tissues of the plant such as the stem tissue. This growth phase is discussed further below and it is within the general skills of the skilled person to identify when a relevant plant is in this growth phase.
  • the term "where the plant is grown under normal natural growth conditions of the plant” relates to an objective of the invention, which is to identify DAHP genes, which are specifically high expressed in stem tissue under such natural growth conditions of the plant (e.g. without specific physical wounding of the plant).
  • Natural growth conditions relates to temperature, humidity and etc that give a normal natural growth of the plant, such as conditions that correspond to conditions for industrial (e.g. agricultural) relevant growth of the plant. It is within the skilled person general knowledge to identify such natural growth conditions.
  • a second aspect of the invention relates to a process for making a plant cell that comprises a stable modification of the genome, wherein the genome modification causes a stable reduction in the amount of mRNA level of a DAHP synthase that is expressed in relatively high amounts in stem tissue of the plant comprising:
  • stable modification of the genome relates to that the genomic modification is stably maintained for generations of the plant cell and also maintained in a plant regenerated from the plant cell.
  • a DAHP synthase in relation to DAHP synthase that is expressed in relatively high amounts in stem tissue shall be understood in accordance with the normal literal meaning of the word "identifiable”.
  • a DAHP synthase is a DAHP synthase that is expressed in relatively high amounts in stem tissue if the DAHP synthase fulfils the screening criteria of the method for identifying a DAHP synthase of the first aspect of the invention and as further described herein.
  • a DAHP synthase that is expressed in relatively high amounts in stem tissue.
  • a DAHP synthase identifiable by the method of the first aspect can not be the DAHP synthase of the plant that has the lowest expression in the stem tissue, but it may be a DAHP synthase that has the second lowest expression in the stem tissue.
  • DAHP synthase genes may be the second lowest expressed DAHP synthase gene.
  • a third aspect of the invention relates to a plant cell that comprises a stable modification of the genome, wherein the genome modification causes a stable reduction in the amount of mRNA level of a DAHP synthase that is expressed in relatively high amounts in stem tissue obtainable by a process for making such a plant cell of the second aspect of the invention and as further described herein.
  • a plant cell of the third aspect that is obtainable by a process for making such a plant cell of the second aspect of the invention will have the described stable genomic modification as a structural characteristic that differentiates it from e.g. the corresponding natural cell. Accordingly, as a product the plant cell as such may be seen as a non-natural cell that is genetically modified and thereby different from e.g. the corresponding natural cell.
  • a fourth aspect of the invention relates to a process for obtaining a plant that comprises a stable modification of the genome, wherein the genome modification causes a stable reduction in the amount of mRNA level of a DAHP synthase that is expressed in relatively high amounts in stem tissue comprising regeneration of the plant from the plant cell of the third aspect of the invention and as further described herein to obtain the plant.
  • a fifth aspect of the invention relates to a plant that comprises a stable modification of the genome, wherein the genome modification causes a stable reduction in the amount of mRNA level of a DAHP synthase that is expressed in relatively high amounts in stem tissue of the plant obtainable by a process for obtaining a plant of the fourth aspect of the invention and as further described herein.
  • the regenerated plant as such, of the fifth aspect will have the described stable genomic modification as a structural characteristic that differentiates it from e.g. the corresponding natural plant. Accordingly, as a product the plant as such may be seen as a non-natural plant that is genetically modified and thereby different from e.g. the corresponding natural plant.
  • the genetically modified plant as described herein may comprise less lignin as compared to the same plant without the stable modification of the genome and this is a an advantage with respect to e.g. improved digestibility of the plant when e.g. used as a feed for an animal.
  • a sixth aspect of the invention relates to use of the plant of the fifth aspect of the invention and as further described herein as a feed for an animal.
  • the forage grass plant may simply be growth on a field where e.g. ruminant animal are present.
  • the plant is e.g. maize, it may be preferred that the maize plant first is grown and then harvested to be used to make an ensilage like feed.
  • Ensilage feed is generally understood as fodder harvested while green and kept succulent by partial fermentation in some way and then used as a concentrated feed source e.g. as tablets.
  • a "chimeric DNA construct” refers to a DNA construct comprising heterogeneous regulatory and coding sequences of a gene.
  • DAHP synthase refers to the enzyme 3-deoxy-7-phosphoheptulonate (DAHP) synthase (EC 2.5.1.54) (Formerly EC 4.1.2.15). It is the first enzyme of the Shikimate pathway. The first seven steps of the Shikimate pathway may also be termed the prechorismate pathway. The Shikimate pathway relates to the ability to synthesize aromatic amino acids de novo in bacteria, fungi, and plants (see figure 1 herein). DAHP synthase catalyzes the reaction:
  • the activity of the enzyme may be determined according to the art as described in (Jones, J. et al, Plant Physiol. (1995) 108: 1413-1421) page 1414, column 2, last paragraph under section "Protein Extraction, DAHP synthase Enzyme Activity."
  • dot refers to plants in which the developing plant has two seed leaves or cotyledons.
  • elongation growth stage of the plant denotes the stage during which culm or stem elongation occurs and is often referred to as jointing. Sub-stages of the elongation stage are defined by the number of nodes that have become either palpable or visible as the result stem elongation. The elongation stage ceases when the inflorescence is enclosed in the uppermost leaf sheath, which is commonly referred to as boot stage. The elongation stage is followed by the reproductive stage, which begins with emergence of the inflorescence and continues through anthesis and fertilization. For further details reference is made to the review article (Moore KJ. et al, "Describing and quantifying growth stages of perennial forage grasses", Agron. J. 83:1073- 5 1077 (1991)).
  • an "endogenous gene” refers to the native gene normally found in its natural location in the genome.
  • encoding and coding refer to the process by which a gene, through the mechanisms of transcription provides the information to a cell from which an mRNA may be transcribed.
  • the mRNA may then in some cases, by the mechanism of translation, be translated into a specific amino acid sequence to produce a protein such as an enzyme. However, it is not always the case. For instance, if the mRNA is anti-sense mRNA there is generally no translation 5 into a protein.
  • Gene refers to a nucleic acid fragment that can be transcribed into a mRNA, including regulatory sequences preceding (5' non-coding) and following (3' non-coding) the coding region. 0
  • lignin refers to hydroxyphenyl (H), guaiacyl(G) and syringyl(S) units found in lignified plant tissue. 5
  • the term "monocot” refers to plants in which the developing plant has only one seed-leaf or cotyledon.
  • operable linked refers to nucleic acid sequences on a single nucleic acid molecule 0 which are associated so that the function of one is affected by the other.
  • plant includes whole plants, portions of plants or plant organs (e. g., roots, stems, leaves, etc.).
  • plant cell denotes a cell, which after proper regeneration (growing) may give rise to a plant.
  • promoter refers to a DNA sequence in a gene, usually upstream (5 1 ) to its coding sequence, which controls the expression of the coding sequence by providing the recognition site for RNA polymerase and other factors required for proper transcription.
  • a promoter may also contain DNA sequences that are involved in the binding of protein factors, which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
  • regulatory sequences refer to nucleotide sequences located upstream (5 1 ), within, and/or downstream (3 1 ) of a coding sequence, which control the transcription and/or expression of the coding sequences in conjunction with the protein biosynthetic apparatus of the cell. These regulatory sequences include promoters, translation leader sequences, transcription termination sequences, and polyadenylation sequences.
  • Transgene or “foreign gene” refers to a gene not normally found in the host organism but one that is introduced by gene transfer.
  • the transgene may be a natural gene from another species than the host or e.g. be a non-natural gene sequence such as e.g. a specifically constructed anti-sense sequence.
  • Transgenic plant or “Transgenic plant cell” denote a plant or a plant cell, which in its genome comprise a transgene.
  • Transformation refers to the transfer of a foreign gene into the genome of a host organism and its genetically stable inheritance.
  • methods of plant transformation include Agrobacterium-mediated transformation and particle-accelerated or “gene gun” transformation technology. See below for further details.
  • Figure 1 Illustration of prior art methods to obtain a monocotyledonous plant of the grass family with reduced amount of lignin, where the art describes transgenic grass plants, wherein the transgenic plant (by use of anti-sense (AS) technology) expressed lower amount of enzymes that catalyzes the last steps in the biosynthesis of lignin.
  • AS anti-sense
  • examples of such enzymes are caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD).
  • COMP caffeic acid O-methyltransferase
  • CAD cinnamyl alcohol dehydrogenase
  • the present invention is based on a strategy that is focussed on decreasing the quantity of basis material (aromatic amino acids) for lignin production in specific relevant tissue (in particular stem and leaf tissue).
  • a further advantage of decreasing the amount of specific DAHP synthases is that the amount of digestible sugar could subsequently increase in the plant.
  • Figure 2 Characterization of total DAHP synthases expression levels in different tissue of tall fescue. The results demonstrated that DAHP synthase mRNA only accumulates to very low levels in tall fescue root tissue whereas slightly higher levels were observed in young and old leaves. In contrast, DAHP synthase mRNA accumulates to a much higher levels stem tissue suggesting that higher amounts of aromatic amino acids are produced in stem tissue. For further details see working example 2 herein.
  • Figure 3 Characterization of specific DAHP synthases expression profiles in different tissue of tall fescue. The results demonstrate that the DAHP synthase 5 accumulates to nearly similar levels in two and six weeks old leaves and in stem tissue. In contrast, only a very week accumulation could be detected in two weeks old roots. A similar expression pattern was observed for DAHP synthase 6 except a significantly lover mRNA accumulation was detected in stem and roots as compared to DAHP synthase 5.
  • DAHP synthase 4 mRNA accumulated to much higher levels in stem tissue of tall fescue than in young leaves and roots where only a very week accumulation could be observed by increasing the number of PCR cycles from 28 cycles for DAHP synthase 4 and 5 to 32 PCR cycles used for comparing expression levels of DAHP synthase 4 and 6.
  • PCR cycles from 28 cycles for DAHP synthase 4 and 5 to 32 PCR cycles used for comparing expression levels of DAHP synthase 4 and 6.
  • the objective of this method is to identify a DAHP synthase relatively highly expressed in stem tissue.
  • the term "relatively” shall be seen in relation to at least one another DAHP synthase of the plant.
  • the method requires identification of at least two different DAHP synthases of the plant. Based on common knowledge and for instance the fact that numerous DAHP synthases DNA sequences are known to the skilled person it is routine work for the skilled person to identify different DAHP synthase gene sequences in a plant of interest.
  • Determination of mRNA levels may e.g. be done by RNA gel blot assays or quantitative PCR. This is routine work for the skilled person. See e.g. working examples herein for an illustrative example in the forage grass tall fescue.
  • a final step of the method relates to comparing the mRNA levels of the DAHP synthases and identify a DAHP synthase wherein the mRNA level of the DAHP synthase in the stem tissue is higher than the mRNA level of a different DAHP synthase of the same plant.
  • an embodiment of the invention relates to step (iv) of the method of the first aspect of the invention, wherein the mRNA level of the DAHP synthase in the stem tissue is at least 1.5 times higher than the mRNA level of a different DAHP synthase of the same plant, more preferably, wherein the mRNA level of the DAHP synthase in the stem tissue is at least 2 times higher than the mRNA level of a different DAHP synthase of the same plant and even more preferably, wherein the mRNA level of the DAHP synthase in the stem tissue is at least 3 times higher than the mRNA level of a different DAHP synthase of the same plant.
  • a preferred DAHP synthase is a DAHP that is expressed in relatively high amounts in stem tissue but not expressed in comparative higher amount in the root tissue.
  • the amount of mRNA of the DAHP synthase in the stem tissue is at least 25% higher than in the root tissue, even more preferably the amount of mRNA of the DAHP synthase in the stem tissue is at least 75% higher than in the root tissue and most preferably the amount of mRNA of the DAHP synthase in the stem tissue is at least 200% higher than in the root tissue. This is preferably analyzed by quantitative PCR as known to the skilled person.
  • the method requires identification of at least two different DAHP synthase genes. However, one may of coerce identify more different DAHP genes and then use all of these to identify the ones that are expressed to highest levels in stem tissue.
  • a preferred embodiment of the invention is, wherein there in step (i) is identified three, four, five, six or more different DAHP synthase genes and the identified DAHP synthase with high mRNA level in the stem tissue is the DAHP synthase with highest mRNA level in the stem tissue.
  • the identified DAHP synthase with high mRNA level in the stem tissue has a mRNA level in root tissue that is not higher than any of the different DAHP synthase used for comparison of the mRNA level in stem tissue.
  • the plant or plant cell may in principle be any plant including trees.
  • the plant may be a dicotyledons (dicot) plant from the family Magnoliopsida.
  • a preferred dicot plant is a dicot plant selected from the group consisting of tobacco, tomato, potato, lucerne, lettuce, cotton, cabbage, mustard and Arabidopsis thaliana.
  • a most preferred dicot plant is a dicot plant selected from the group consisting of tomato, potato and lucerne.
  • the plant is a tree.
  • a preferred tree is a plant selected from the family Magnoliopsida such as, the family Liliopsida such as palm trees or from the group of cornifers such as pine and spruce trees.
  • a more preferred plant is a monocotyledonous plant, wherein a most preferred plant is a plant or a plant cell of the grass family.
  • Gramss family should herein be understood according to the art. It denotes a plant family (Poaceae, formerly Graminaeae) of monocotyledonous mostly herbaceous plants with jointed stems, slender sheathing leaves, and flowers borne in spikelets of bracts.
  • the grass family includes plants such as a grass (e.g. a forage grass such as tall fescue), the cereals such as maize, wheat, oats, barley, rye, corn, sorghum, rice, triticale or millet and bamboo.
  • the plant or plant cell of the grass family is a plant or plant cell selected from the group consisting of: a grass of the family Poaceae (e.g. a forage grass such tall fescue), and a cereal such as a maize, a wheat, an oat, a barley, a rye, a corn, a sorghum, a rice, a triticale and a millet.
  • a grass of the family Poaceae e.g. a forage grass such tall fescue
  • a cereal such as a maize, a wheat, an oat, a barley, a rye, a corn, a sorghum, a rice, a triticale and a millet.
  • a preferred plant is maize or rice and a particular preferred plant is a forage grass such as in particular the forage grass tall fescue (Festuca arundinaced).
  • Preferred grass species include a ryegrass (Lolium species) or fescue (Festuca species).
  • Preferred rice species include the genus Oryza.
  • Preferred wheat species include the genus Triticum
  • Preferred oat species include the genus Avena.
  • Preferred maize species include the genus Zea.
  • a process for making a plant cell with a genome modification :
  • An essential step of the process for making a plant cell with a genome modification of the second aspect of the invention is step (i) that relates to: modifying the genome of the cell in a way that, after regeneration of the cell to get a plant, causes a stable reduction in the amount of mRNA level of the DAHP synthase, wherein the DAHP synthase is identifiable by a method for identifying a DAHP synthase that is expressed in relatively high amounts in stem tissue of a plant of the first aspect and as further described herein.
  • the herein described DAHP synthase that is specifically expressed in the lignin producing stem tissue may also be relatively highly expressed in other tissue such as preferably in leaf, spikelts, in the husk surrounding the seed or in the ear of maize.
  • the genome modification may be done in different ways as known to the skilled person.
  • a preferred way is by use of the so-called anti-sense technology or or RNA interference technology.
  • a preferred embodiment of the invention is, wherein the modification of the genome is done by anti-sense technology comprising incorporation of a chimeric DNA construct into the genome, wherein the chimeric DNA construct comprises a heterogeneous regulatory sequence operable linked in anti-sense orientation to a coding sequence of the DAHP gene making the cell capable of translating an anti-sense mRNA fragment that within the cell can hybridize to the mRNA of the DAHP synthase. 5
  • anti-sense technology is well described in the art and is also further described herein.
  • transposable elements 10 insertional mutagenesis by transposable elements or T-DNA tagging has been used successfully.
  • the DNA causing gene disruption marks the respective locus and subsequently can be used as a molecular probe to isolate a defined gene.
  • Transposable elements such as Mu, AdDs and En/Spm of Zea mays, Tarn of Antirrhinum majus, and dTph of Petunia hybrida, have been applied for gene tagging in their native hosts, and AdDs and En/Spm also
  • TILLING Targeting induced local lesions in genomes
  • the DAHP synthase that is expressed in relatively higher amount in the stem tissue is preferably down regulated in the leaf and stem tissue.
  • the DAHP may be present in normal amount in other tissues of the plant such as root, flower etc.
  • Such a specific down regulation in leaf and/or stem tissue may be achieved by use of adequate regulatory sequences that directs transcription preferably to lignifying leaf and stem tissue.
  • regulatory sequences are known in the art and suitable examples are the Adhi promoter from maize, which directs expression to the vascular tissues and lignifying sclerenchyma (Piquemal et al 2002. Plant Physiol. 130, 1675-1685), the CCR promoter from Lolium perenne (patent WO 02/50294 Al) or the 4CL, OMT and CAD promoters from Lolium perenne (Patent WO 01/95702 Al).
  • the genomic modification causes a stable at least 2 times reduction in the amount of mRNA level of the DAHP synthase that is expressed in relatively high amounts in stem tissue as compared the corresponding regenerated plant without the genomic modification, more preferably the genomic modification causes a stable at least 4 times reduction, even more preferably a stable at least 6 time reduction and most preferably the genomic modification causes a stable at least 10 times reduction in the amount of mRNA level of the DAHP synthase that is expressed in relatively high amounts in stem tissue as compared the corresponding regenerated plant without the genomic modification.
  • DAHP synthase specifically expressed in the lignin producing stem tissue as described herein is a DAHP synthase wherein the amount of mRNA of the DAHP synthase in the stem tissue is at least 10% higher than in the root tissue. More preferably, the amount of mRNA of the DAHP synthase in the stem tissue is at least 25% higher than in the root tissue, even more preferably the amount of mRNA of the DAHP synthase in the stem tissue is at least 75% higher than in the root tissue and most preferably the amount of mRNA of the DAHP synthase in the stem tissue is at least 200% higher than in the root tissue. This is preferably analyzed by quantitative PCR as known to the skilled person.
  • One advantage of targeting a DAHP synthase specifically expressed in the lignin producing stem tissue as described herein is that one may get a plant where this DAHP is mainly targeted and other DAHP synthases of the plant functions in a general normal way.
  • a preferred embodiment of the invention is, wherein the genomic modification does not significantly affect the natural mRNA expression level of at least one other DAHP synthase of the regenerated plant.
  • a constitutive promoter to drive the expression of e.g. the antisense sequence specific for the DAHP synthase of interest.
  • the genomic modification essentially only affect the mRNA expression level of the DAHP synthase that is expressed in relatively high amounts in stem tissue as described herein.
  • An advantage of mainly down regulating the specific DAHP that are highly expressed in stem tissue is that other DAHP genes of the plants functions in a normal way. If one down regulates all DAHP genes of the plant there is a greater risk to get a negative unwanted influence on the e.g. growth and development of the plant.
  • a tissue specific promoter mainly active in only lignifying tissue.
  • antisense technology one could here use a sequence common to all DAHP synthase genes one wants to affect.
  • RNA interference anti-sense technology where short (around 15-35 base pairs) anti-sense sequences are used.
  • the short anti-sense sequences are highly specific and hybridize only to a specific target mRNA of interest. This technology is further described in working examples herein.
  • DAHPSl DAHP synthase
  • SEQ ID NO 1 is genomic DNA sequence of DAHPSl (termed “DAHPS 1-gDNA”);
  • SEQ ID NO 2 is translated amino acid sequence of DAHPSl (termed “DAHPS 1-aa”);
  • SEQ ID NO 3 is genomic DNA sequence of DAHPS2 (termed “DAHPS2-gDNA”);
  • SEQ ID NO 4 is translated amino acid sequence of DAHPS2 (termed “DAHPS2-aa”);
  • SEQ ID NO 5 is genomic DNA sequence of DAHPS3 (termed “DAHPS3-gDNA”);
  • SEQ ID NO 6 is translated amino acid sequence of DAHPS3 (termed “DAHPS3-aa”);
  • SEQ ID NO 7 is cDNA sequence of DAHPS4 (termed “DAHPS4-cDNA”);
  • SEQ ID NO 8 is translated amino acid sequence of DAHPS4 (termed “DAHPS5-aa”);
  • SEQ ID NO 9 is cDNA sequence of DAHPS5 (termed “DAHPS5-cDNA”);
  • SEQ ID NO 10 is translated amino acid sequence of DAHPS5 (termed “DAHPS5-aa”);
  • SEQ ID NO 11 is cDNA sequence of DAHPS6 (termed “DAHPS6-cDNA”);
  • SEQ ID NO 12 is translated amino acid sequence of DAHPS6 (termed “DAHPS6-aa”);
  • SEQ ID NO 13 is cDNA sequence of DAHPS7 (termed “DAHPS7-cDNA”);
  • SEQ ID NO 14 is translated amino acid sequence of DAHPS7 (termed “DAHPS7-aa”);
  • SEQ ID NO 15 is cDNA sequence of DAHPS8 (termed “DAHPS8-cDNA”);
  • SEQ ID NO 16 is translated amino acid sequence of DAHPS8 (termed “DAHPS8-aa”);
  • SEQ ID NO 17 is cDNA sequence of DAHPS9 (termed “DAHPS9-cDNA”); SEQ ID NO 18 is translated amino acid sequence of DAHPS9 (termed “DAHPS9-aa”);
  • SEQ ID NO 19 is cDNA sequence of DAHPSlO (termed “DAHPS 10-cDNA”);
  • SEQ ID NO 20 is translated amino acid sequence of DAHPSlO (termed “DAHPS 10-aa”);
  • a DAHP synthase that is expressed in relatively high amounts in stem tissue of a plant is a DAHP synthase gene comprising a DNA sequence selected from at least one of the ten groups of DNA sequences consisting of:
  • a DNA sequence wherein the DNA sequence is selected from the group consisting of: (a) the DNA sequence shown in positions 1-3075 in SEQ ID NO 1 (termed “DAHPS 1-gDNA”); (b) a DNA sequence that encodes a polypeptide, comprised within a longer polypeptide having DAHPS activity, that is at least 90% identical to the polypeptide sequence shown in positions 1- 337 of SEQ ID NO 2 (termed "DAHPSl-aa”); (c) a DNA sequence which is at least 90% identical to the DNA sequence of (a);
  • DNA sequence wherein the DNA sequence is selected from the group consisting of: (a) the DNA sequence shown in positions 1-2989 in SEQ ID NO 3 (termed “DAHPS2-gDNA”); (b) a DNA sequence that encodes a polypeptide, comprised within a longer polypeptide having DAHPS activity, that is at least 90% identical to the polypeptide sequence shown in positions 1- 337 of SEQ ID NO 4 (termed "DAHPS2-aa”);
  • DNA sequence wherein the DNA sequence is selected from the group consisting of:
  • DNA sequence wherein the DNA sequence is selected from the group consisting of: (a) the DNA sequence shown in positions 1-1011 in SEQ ID NO 7 (termed “DAHPS4-cDNA”);
  • V a DNA sequence, wherein the DNA sequence is selected from the group consisting of:
  • DNA sequence wherein the DNA sequence is selected from the group consisting of: (a) the DNA sequence shown in positions 1-1011 in SEQ ID NO 11 (termed “DAHPS6- cDNA”);
  • DAHPS activity that is at least 90% identical to the polypeptide sequence shown in positions 1-
  • DNA sequence wherein the DNA sequence is selected from the group consisting of:
  • DNA sequence wherein the DNA sequence is selected from the group consisting of:
  • Villi a DNA sequence, wherein the DNA sequence is selected from the group consisting of: (a) the DNA sequence shown in positions 1-1466 (preferably positions 1-1185) in SEQ ID NO 17 (termed “DAHPS9-CDNA”);
  • X a DNA sequence, wherein the DNA sequence is selected from the group consisting of:
  • a DNA sequence that encodes a polypeptide of (b) of group / is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 2, more preferably at least 30 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 2.
  • a DNA sequence that encodes a polypeptide of (b) of group // is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 4, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 4.
  • a DNA sequence that encodes a polypeptide of (b) of group /// is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 6, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 6.
  • a DNA sequence that encodes a polypeptide of (b) of group IV is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 8, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 8.
  • a DNA sequence that encodes a polypeptide of (b) of group V is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 10, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 10.
  • a DNA sequence that encodes a polypeptide of (b) of group VI is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 12, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 12.
  • a DNA sequence that encodes a polypeptide of (b) of group VII is preferably at least 95% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 14, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 14.
  • a DNA sequence that encodes a polypeptide of (b) of group VIII is preferably at least 95% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 16, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 16.
  • a DNA sequence that encodes a polypeptide of (b) of group Villi is preferably at least 95% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 18, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 18.
  • a DNA sequence that encodes a polypeptide of (b) of group X is preferably at least 95% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 20, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 20.
  • a DNA sequence of (c) is preferably at least 95% identical to the DNA sequence of (a), more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence of (c) is preferably at least 99% identical to the to the DNA sequence of (a).
  • hybridization of (d) is done at very high stringency.
  • This embodiment with respect of the ten groups of DNA sequences above, is particular relevant, wherein the plant cell is a forage grass cell, in particular a tall fescue cell.
  • the final step (ii) of this process relates to isolating the cell with the stable modification of the genome.
  • DAHPS 1-gDNA a DNA sequence that encodes a polypeptide, comprised within a longer polypeptide having DAHPS activity, that is at least 90% identical to the polypeptide sequence shown in positions 1- 337 of SEQ ID NO 2 (termed “DAHPSl-aa”);
  • DNA sequence is selected from the group consisting of: (a) the DNA sequence shown in positions 1-2989 in SEQ ID NO 3 (termed “DAHPS2-gDNA”); (b) a DNA sequence that encodes a polypeptide, comprised within a longer polypeptide having DAHPS activity, that is at least 90% identical to the polypeptide sequence shown in positions 1- 337 of SEQ ID NO 4 (termed "DAHPS2-aa”);
  • DAHPS activity that is at least 90% identical to the polypeptide sequence shown in positions 1-
  • Villi An isolated DNA sequence, wherein the DNA sequence is selected from the group consisting of:
  • X An isolated DNA sequence, wherein the DNA sequence is selected from the group consisting of:
  • DHPS 10-cDNA 19 (termed “DAHPS 10-cDNA”); (b) a DNA sequence that encodes a polypeptide, comprised within a longer polypeptide having
  • DAHPS activity that is at least 90% identical to the polypeptide sequence shown in positions 1-
  • An isolated DNA sequence refers to a DNA sequence isolated (cloned) in accordance with standard cloning procedures used in genetic engineering to relocate a segment of DNA from its natural location to a different site where it will be reproduced.
  • the cloning process involves excision and isolation of the desired DNA segment, insertion of the piece of DNA into the vector molecule and incorporation of the recombinant vector into a cell where multiple copies or clones of the DNA segment will be replicated.
  • a DNA sequence that encodes a polypeptide of (b) of group / is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 2, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 2.
  • a DNA sequence that encodes a polypeptide of (b) of group // is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 4, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 4.
  • a DNA sequence that encodes a polypeptide of (b) of group /// is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 6, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 6.
  • a DNA sequence that encodes a polypeptide of (b) of group IV is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 8, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 8.
  • a DNA sequence that encodes a polypeptide of (b) of group V is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 10, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 10.
  • a DNA sequence that encodes a polypeptide of (b) of group VI is preferably at least 95% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 12, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-337 of SEQ ID NO 12.
  • a DNA sequence that encodes a polypeptide of (b) of group VII is preferably at least 95% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 14, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 14.
  • a DNA sequence that encodes a polypeptide of (b) of group VIII is preferably at least 95% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 16, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 16.
  • a DNA sequence that encodes a polypeptide of (b) of group Villi is preferably at least 95% identical to the polypeptide sequence shown in positions 1 -394 of SEQ ID NO 18, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 18.
  • a DNA sequence that encodes a polypeptide of (b) of group X is preferably at least 95% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 20, more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence that encodes a polypeptide of (b) is preferably at least 99% identical to the polypeptide sequence shown in positions 1-394 of SEQ ID NO 20.
  • a DNA sequence of (c) is preferably at least 95% identical to the DNA sequence of (a), more preferably at least 97% identical, even more preferably at least 98% identical and most preferably a DNA sequence of (c) is preferably at least 99% identical to the to the DNA sequence of (a).
  • hybridization of (d) is done at very high stringency.
  • a plant obtainable by use of the genomic modified plant cell :
  • genomic modified plant cell Once having made and isolated the genomic modified plant cell as described herein it is routine to use this cell to regenerate the plant from the plant cell to obtain the plant as such.
  • Regeneration relates essentially to adequate growth of the plant cell to get the plant as such.
  • an advantage of reducing the amount of the stem tissue specific DAHP as described herein is that the plant comprises less lignin.
  • an embodiment of the invention relates to, wherein the plant during the elongation growth stage comprises less lignin as compared to the same plant without the stable modification of the genome that causes a stable reduction in the amount of mRNA level of a DAHP synthase that is expressed in relatively high amounts in stem tissue.
  • the amount of lignin is the amount of lignin determined by use of the so-called Klason method. This is described in details in the reference (Knudsen, Animal Feed Science Technology 67 (1997) 319- 338).
  • the amount of lignin is determined by taking stem tissues from 10 individual plants during the elongation growth stage. The amount of lignin is measured and the amount of lignin in the stem tissue is then understood to be the average amount of the 10 individual plants.
  • a transgenic plant of the invention as described herein also comprises less lignin in the leave tissues.
  • the animal is a ruminant animal such as a cattle, cow, goat or horse.
  • a DNA sequence i.e., of transforming
  • Such methods include those based on transformation vectors based on the Ti and Ri plasmids of Agrobacterium spp. It is particularly preferred to use the binary type of these vectors.
  • Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants.
  • Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs [see EPO publication 0 295 959 A2], techniques of electroporation [see Fromm et al.
  • RNA constructs of interest such a chimeric DNA construct of interest.
  • the promoter in the constructs suitably provides for expression of the linked DNA segment.
  • the promoter can also be inducible so that gene expression can be turned on or off by an exogenously added agent. It may also be preferable to combine the desired DNA segment with a promoter that provides tissue specific expression or developmentally regulated gene expression in plants.
  • the promoter may be selected from promoters known to operate in plants. Suitable examples are described in US2002/0138870A1 section [0074] and include e.g., CaMV35S, GPAL2, GPAL3 and endogenous plant promoter controlling expression of the enzyme of interest.
  • a constitutive promoter such as the CaMV35S promoter, or CaMV 19S can be used to drive the expression of the transgenes in all tissue types in a target plant.
  • Other promoters are nos, Adh, sucrose synthase, ⁇ -tubulin, ubiquitin, actin, cab, PEPCase or those associate with the R gene complex.
  • use of a tissue specific promoter permits functions to be controlled more selectively.
  • the use of a tissue-specific promoter has the advantage that the desired protein is only produced in the tissue in which its action is required.
  • selectable markers include, a bar gene which codes for bialaphos resistance; a gene which encodes an altered EPSP synthase protein thus conferring glyphosate resistance; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance tobromoxynil.
  • Screenable markers that may be employed include a ⁇ -glucuronidase or uidA gene (GUS) which encodes an enzyme for which various chromogenic substrates are known; a ⁇ -lactamase gene, which encodes an enzyme for which various chromogenic substrates are known. Examples of other selectable or screenable markers may be found e.g. in table 1 of WOO 1/27241.
  • the DNA sequence identity referred to herein is determined as the degree of identity between two sequences indicating a deviation of the first sequence from the second.
  • NCBI National Center for Biotechnology Information
  • a preferred computer homology search program is a "Standard nucleotide-nucleotide BLAST [blastn]" search as specified, at the filing date of the present application, at the NCBI Internet site with setting filter: Low complexity; Expect: 10, Word Size: 11.
  • the reference sequence is introduced into the program and the program identifies fragments of another sequence (e.g. a published sequence) together with the identity percentage to a corresponding fragment of the reference sequence.
  • a specific reference sequence e.g. DNA sequence 1-1011 in SEQ ID NO 7 - termed "DAHPS4-cDNA"
  • said another sequence should have a length which is comparable to the reference sequence. For instance, if the length of the reference sequence is 1000 bp a comparable length of the other sequence could e.g. be from 800 - 1200 bp.
  • the hybridization referred to above is intended to comprise an analogous DNA sequence which hybridizes to a double-stranded DNA probe.
  • Suitable experimental conditions for determining hybridization at low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involve presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al.
  • Example 1 Isolation of Tall fescue DAHP synthase genes 0
  • DAHP synthase genes from Tall fescue total RNA was prepared from plants grown in a green house at a temperature of 21 0 C with 12 hrs of daylight. Tissue was harvested from two weeks old roots, from two weeks old leaves, from 6 weeks old leaves and from 6 weeks old stem tissue and RNA was isolated using the "Total RNA Isolation System" from Promega.
  • DAHP synthases from rice and Arabidopsis was extracted from public databases and aligned using the program AlignX from the Vector NTI suite 9.0 software packaged (Infomax). 5 Subsequently, sets of degenerate primers were synthesized based on DNA motifs showing the highest degree of identity.
  • a and B were combinations of ABJ49 + ABJ53 or ABJ49 + ABJ32 respectively.
  • the PCR reactions were purified using the High Pure PCR Product Purification Kit (Roche) and 3 ⁇ l of the purified reactions were used for TOPO Blunt Cloning into the pCR-Bluntll-TOPO 30 plasmid. The resulting colonies were sequenced and sequence analysis revealed that three different partial DAHPS synthase genes had been isolated. These genes were designated DAHPS4, DAHPS5 and DAHPS6.
  • the PCR reactions were heated to 94°C for 2 min and run for 30 cycles at 94°C for 30 sec, 60 0 C for 30 sec followed by 72°C for 60 sec.
  • Example 2 Characterization of total DAHP synthases expression levels in different tissue of tallfescue
  • RNA gel blot analysis was performed. First, total RNA was isolated from two weeks old roots, from two weeks old leaves, from 6 weeks old leaves and from 6 weeks old stem tissue using a "Total RNA Isolation System" from Promega. Next, 20 ⁇ g total RNA was fractionated on a 1% formaldehyde denaturing agarose gel and transferred onto a nylon Hybond N membrane from Amersham Biosience according to the instructions of the manufacturer. Then, DNA fragments of DAHP synthases 4, 5, 6, 7, 8, 9 and 10 were isolated by liberating the inserts using EcoRI restriction enzyme digestion followed by gel purification.
  • the purified DNA fragments were mixed in equal amounts and hybridization and probes were labelled with Oc 32 P- dCTP using a Random primed DNA Labelling Kit from Roche.
  • As a control a 258 bp fragment of the 5.8 rRNA were isolated by PCR according to the procedure described by L ⁇ cher et al 2000. Plant Physiol. VoI 124, 1217-1227.
  • DAHP synthase mRNA only accumulates to very low levels in tall fescue root tissue whereas slightly higher levels were observed in young and old leaves. In contrast, DAHP synthase mRNA accumulates to much higher levels stem tissue suggesting that higher amounts of aromatic amino acids are produced in stem tissue.
  • DAHP synthase 5 accumulates to nearly similar levels in two and six weeks old leaves and in stem tissue. In contrast, only a very week accumulation could be detected in two weeks old roots. A similar expression pattern was observed for DAHP synthase 6 except a significantly lover mRNA accumulation was detected in stem and roots as compared to DAHP synthase 5.
  • DAHP synthase 4 mRNA accumulated to much higher levels in stem tissue of tall fescue than in young leaves and roots where only a very week accumulation could be observed by increasing the number of PCR cycles from 28 cycles for DAHP synthase 4 and 5 to 32 PCR cycles used for comparing expression levels of DAHP synthase 4 and 6.
  • DAHP synthase 4 was expressed to 2.5 fold higher levels than DAHP synthase 5 and to 4 fold higher levels than DAHP synthase 6 in 6 weeks old tall fescue stem tissue.
  • DAHP synthase activity the strong constitutive maize ubiqutin promoter including it's first intron followed by the pea terminator signal E9 is inserted into the EcoRI-BamHI sites of the plant transformation vector pCAMBIA1300.
  • This vector is designated pCAMBIA-ubi-E9.
  • DAHP synthase 4, 5 and 6 will be liberated from then- respective plasmid vectors by restriction enzyme digestion by BamHI+Xbal and inserted into the pCAMBIA-ubi-E9 vector digested by the same enzymes such that DAHP synthase 4, 5 and 6 are placed in their antisense orientation.
  • DAHP synthase 4, 5 and 6 After transformation into tall fescue, maize and rice such vectors result in down regulation of all DAHP synthases in tissue wherein the ubiqutin promoter is active.
  • Adhl promoter that is active in vascular tissue and lignifying sclerenchyma (Piquemal J. et al, Plant Physiol, December 2002, Vol. 130, pp. 1675-1685) is used to drive antisense expression of DAHP synthase 4, 5 and 6.
  • Exchanging the ubiqutin promoter in pCAMBLA-ubi-E9 for the Adhl promoter by restriction enzyme digestion with EcoRI+BamHI makes this vector, designated pC AMBIA- Adhl-E9.
  • DAHP synthase 4, 5 and 6 is liberated from their respective plasmid vectors by restriction enzyme digestion with BamHI+Xbal and inserted into the pCAMBIA-AdhI-E9 vector digested by the same enzymes such that DAHP synthase 4, 5 and 6 are placed in their antisense orientation.
  • restriction enzyme digestion with BamHI+Xbal restriction enzyme digestion with BamHI+Xbal
  • pCAMBIA-AdhI-E9 vector digested by the same enzymes such that DAHP synthase 4, 5 and 6 are placed in their antisense orientation.
  • the ryegrass OMT promoter described in patent WO 01/95702 Al will be chemical synthesized and exchanged with the Adhl promoter of plasmid pCAMBIA-AdhI-E9 resulting in the plasmid designated pCAMBIA-OMT-E9.
  • DAHP synthase 4, 5 and 6 is liberated from their respective plasmid vectors by restriction enzyme digestion with BamHI+Xbal and inserted into plasmid pCAMBIA-OMT-E9. Since the OMT gene is a key step in production of precursors for lignin biosynthesis these constructs result in down regulation of all DAHP synthases in tissue undergoing lignification.
  • DAHP synthases like DAHP synthase 4 that are expressed in tissue undergoing lignification
  • DAHP synthases can be specifically down regulated by the use of the RNAi technology.
  • short RNAi fragments of 100-200 bp sequences corresponding to DAHP synthases, specifically expressed in root and leaf tissue undergoing lignification, are chemically synthesized with and intervening intron like sequence as spacer.
  • the resulting DNA fragments are inserted into plant transformation vectors such as pC AMBIA- ubi-E9, pCAMBIA-AdhI-E9 and pCAMBIA-OMT-E9 thereby creating expression cassettes driven by promoter sequences that either constitutively down regulates specific DAHP synthases or which directs the down regulation to specific cells undergoing lignification.
  • plant transformation vectors such as pC AMBIA- ubi-E9, pCAMBIA-AdhI-E9 and pCAMBIA-OMT-E9 thereby creating expression cassettes driven by promoter sequences that either constitutively down regulates specific DAHP synthases or which directs the down regulation to specific cells undergoing lignification.
  • Such a RNAi strategy which is based on down regulating specific DAHP synthase, ensures that DAHP synthase necessary for production of aromatic amino acids in cells not undergoing lignification is still normally active. Thereby, negative effects of down regulating
  • constructs described above are designed such that they can be directly used for transformation of all plants by agrobacterium mediated transformation techniques. They can also be used directly for transformation by particle bombardment or DNA fragments containing the promoter-antisense cassettes can be isolated from the corresponding plasmids and used alone or in combination with other DNA fragments carrying selectable markers for subsequent identification of the transgenic plants.
  • Promoter regions from the CCR gene from Lolium perenne (patent WO 02/50294 Al) and from the maize ubiquitin gene were amplified by PCR using primers CCR-for 5'- GCGGCCGCCCCTCCCCACAGAAAAGACATCCC-S' (SEQ ID 33) and CCR-rev 5'- CGCGGATCCGAATTCGTTTAAACTGTCGCTCTTACGGTACTACTG-S' (SEQ ID 34) Ubil-for 5'-GTGGCGGCCGCTAATGAGCATTGCATGTCTAAG-S' (SEQ ID 35) and Ubil- rev 5'- TTCGTTTAAACCATTGAAGCGGAGGTGCCGACGGG-S' (SEQ ID 36).
  • the resulting DNA fragment of the CCR promoter was digested with Notl and BamHI and inserted into the pBS(SK " ) plasmid carrying the E9 terminator, designated pBS(SK " )CCR-E9.
  • the pBS(SK " )CCR-E9 vector was digested with Notl and Pmel and the PCR fragment of the maize ubiquitin promoter was inserted into this vector after it had been digested with the same enzymes thereby creating the pBS(SK " )Ubil-E9 plasmid.
  • pBS(SIC)Ubil-antiDAHPS4-E9 Before plasmid pBS(SIC)Ubil-antiDAHPS4-E9, pBS(SK-)Ubil-antiDAHPSall-E9 and pBS(SK " )CCR-antiDAHPSall-E9 were transform into Tall fescue, linear fragments of the promoter- DAHPS-terminator cassette were generated by digestion the plasmids with Notl and Kpnl followed by gel purification to isolate the pure DNA fragments. Afterwards, these fragments were inserted into the pGreenll vector digested with the same enzymes.
  • Tall fescue transformation was subsequently, performed both with linear plasmid free DNA fragments only containing the Ubil-antiDAHPS4-E9, Ubil-antiDAHPSall-E9 and the CCR-antiDAHPSall-E9 antisense expression cassettes and with the T-DNA plasmids pGreenII-Ubil-antiDAHPS4-E9 pGreenII-Ubil-antiDAHPSall-E9 and the pGreenII-CCR-antiDAHPSall-E9.
  • the transformation experiments are performed using a helium driven particle delivery system. DNA constructs are coated onto 1.0 ⁇ m gold particles and delivered into the suspension cells. Selection of stable transformed colonies are performed by transferring bombarded cells onto a solid subculture medium followed by transfer to selection medium after 2 weeks before regeneration of transgenic plantlets. Regenerated shoots are transferred to rooting medium for four to six weeks before the rooted plants are transferred to soil.
  • Lys Ser Arg lie Pro Asp Pro GIn Arg Met lie Arg Ala Tyr Thr GIn
  • GIu Lys Ser Arg lie Pro Asp Pro GIn Arg Met lie Arg Ala Tyr Thr 80 85 90 95 cag teg get gcc acg etc aac etc etc cgc get ttc gcc atg gga ggg 450 GIn Ser Ala Ala Thr Leu Asn Leu Leu Arg Ala Phe Ala Met GIy GIy 100 105 110 tat get gcc atg cag egg gtc ace cag tgg aac etc gat ttc act gaa 498 Tyr Ala Ala Met GIn Arg VaI Thr GIn Trp Asn Leu Asp Phe Thr GIu 115 120 125 aac age gag cag ggt gac ag gtgaaacat cttctatgca ctttctgtgt 548 Asn Ser GIu GIn GIy Asp Arg 130 tttc
  • Asp Ser lie Leu Ala GIu VaI Arg Ala Phe Phe Asp VaI His GIu GIn 305 310 315 320
  • GIu lie Leu Asn Pro Thr Asn Lys Ala GIy Arg lie Thr lie lie Thr
  • Asp Ser lie Leu Ala GIu VaI Arg Ala Phe Phe Asp VaI His GIu GIn 305 310 315 320
  • Asp Ser lie Met Asn GIu VaI Arg Ala Phe Phe Asp VaI His Asp GIn 305 310 315 320
  • GIu GIy Ser His Pro GIy GIy lie His Leu GIu Met Thr GIy GIn Asn
  • Asp Arg lie Leu Ala GIu VaI Arg Ala Phe Phe Asp VaI His GIu GIn 305 310 315 320
  • GIu Ser Phe Lys GIu Phe Asn GIy Asn Asn lie Arg Asp Thr Phe Arg 1 5 10 15 gtc etc etc cag atg tec gee gta etc ace ttc ggc ggc cag atg ccc 96 VaI Leu Leu GIn Met Ser Ala VaI Leu Thr Phe GIy GIy GIn Met Pro 20 25 30 gtc ate aag gtt ggg agg atg gcc ggc cag ttc gcg aag ccg agg teg 144
  • Asp Asn lie Asn GIy Asp Ala Phe Asn GIu Lys Ser Arg lie Pro Asp
  • GIn lie VaI Thr Trp lie Thr Asp Pro Met His GIy Asn Thr lie Lys 275 280 285
  • GIn lie VaI Thr Trp lie Thr Asp Pro Met His GIy Asn Thr lie Lys 275 280 285
  • GIu Ser Phe Lys GIu Phe Asn GIy Asn Asn lie Arg Asp Thr Phe Arg 1 5 10 15 gtc etc etc cag atg tec gee gtc etc ace ttc ggc ggc cag atg cec 96 VaI Leu Leu GIn Met Ser Ala VaI Leu Thr Phe GIy GIy GIn Met Pro 20 25 30 gtc ate aag gtt ggg aga atg gee ggc cag ttc gcg aag ccg agg teg 144 VaI lie Lys VaI GIy Arg Met Ala GIy GIn Phe Ala Lys Pro Arg Ser 35 40 45 gac aac ttc gag gtc aag gac gga gtg aag eta ccc age tac aga ggg 192 Asp Asn Phe GIu Va
  • Pro GIn Arg Met lie Arg Ala Tyr Thr GIn Ser Ala Ala Thr Leu Asn 85 90 95 etc etc cgc get ttc gee atg gga ggc tat get gee atg cag egg gtc 336 Leu Leu Arg Ala Phe Ala Met GIy GIy Tyr Ala Ala Met GIn Arg VaI 100 105 110 ace cag tgg aac etc gat ttc ace gaa aac age gag cag ggt gac agg 384 Thr GIn Trp Asn Leu Asp Phe Thr GIu Asn Ser GIu GIn GIy Asp Arg 115 120 125 tac cgt gaa ttg gca cac agg gtt gat gaa gcc ctt ggc ttc atg tct 432 Tyr Arg GIu Leu Ala His Arg VaI Asp GIu Ala
  • GIn lie VaI Thr Trp lie Thr Asp Pro Met His GIy Asn Thr lie Lys
  • 280 285 get cct tgt ggt eta aag aca cgc ccc ttt gac tct att ctg get gag 912 Ala Pro Cys GIy Leu Lys Thr Arg Pro Phe Asp Ser lie Leu Ala GIu 290 295 300 gtc agg gca ttc ttc gat gtt cat gag caa gaa ggg age cac gca gga 960 VaI Arg Ala Phe Phe Asp VaI His GIu GIn GIu GIy Ser His Ala GIy 305 310 315 320 ggt gtc cac etc gag atg act ggg cag aac gtg aca gag tgc ate ggt 1008 GIy VaI His Leu GIu Met Thr GIy GIn Asn VaI Thr GIu Cys lie GIy 325
  • Asp Asn lie Asn GIy Asp Ala Phe Asn GIu Lys Ser Arg lie Pro Asp 20 65 70 75 80
  • GIn lie VaI Thr Trp lie Thr Asp Pro Met His GIy Asn Thr lie Lys 275 280 285
  • GIn lie VaI Thr Trp lie Thr Asp Pro Met His GIy Asn Thr lie Lys
  • Asp Asn lie Asn GIy Asp Ala Phe Asp VaI Lys Ser Arg Thr Pro Asp 65 70 75 80
  • Ala Ala GIy Leu GIy lie Asp His Pro lie Met Thr Thr Thr Asp Phe 145 150 155 160
  • Phe Leu Arg GIy lie Ala Asn Pro Leu GIy lie Lys VaI Ser Asp Lys 210 215 220
  • GIn lie VaI Thr Trp lie Thr Asp Pro Met His GIy Asn Thr lie Lys 275 280 285
  • GIy lie His Leu GIu Met Thr GIy GIn Asn VaI Thr GIu Cys lie GIy
  • PCR primer ⁇ 400> 34 cgcggatccg aattcgttta aactgtcgct cttacggtac tactg 45

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  • Health & Medical Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
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  • Wood Science & Technology (AREA)
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  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur une plante ou cellule végétale comprenant une modification stable du génome, cette modification du génome entraînant une réduction stable dans la quantité du taux d'ARNm d'une synthase de DAPH qui est exprimée dans des quantités relativement élevées dans le tissu de la tige de la plante. La plante peut avoir une quantité réduite de lignine.
EP05789584A 2004-08-26 2005-08-23 Plante a teneur en lignine reduite par modulation de l'expression genique de dahps Withdrawn EP1802768A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP05789584A EP1802768A2 (fr) 2004-08-26 2005-08-23 Plante a teneur en lignine reduite par modulation de l'expression genique de dahps

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP04104096 2004-08-26
PCT/EP2005/054129 WO2006021558A2 (fr) 2004-08-26 2005-08-23 Plante a teneur en lignine reduite par modulation de l'expression genique de dahps
EP05789584A EP1802768A2 (fr) 2004-08-26 2005-08-23 Plante a teneur en lignine reduite par modulation de l'expression genique de dahps

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EP1802768A2 true EP1802768A2 (fr) 2007-07-04

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US8664475B2 (en) * 2007-09-18 2014-03-04 Basf Plant Science Gmbh Plants with increased yield
WO2012004795A1 (fr) * 2010-07-06 2012-01-12 Yeda Research And Development Co. Ltd. Plantes transgéniques ayant une activité de dahp synthase altérée
CN109354617B (zh) 2018-12-10 2021-09-21 中国科学院东北地理与农业生态研究所 水稻环境条件性致死突变基因osesl1及其编码蛋白和应用
CN111454955B (zh) * 2020-04-27 2023-05-12 广西壮族自治区林业科学研究院 源于尾叶桉CAD基因序列的RNAi片段及其应用
WO2023108018A1 (fr) * 2021-12-07 2023-06-15 Wisconsin Alumni Research Foundation Mutations ponctuelles permettant de stimuler la production d'acides aminés aromatiques et l'assimilation de co2 chez la plante

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US6410826B1 (en) * 1998-06-25 2002-06-25 The Regents Of The University Of California Reduction of lignin biosynthesis in transgenic plants
WO2003000898A1 (fr) * 2001-06-22 2003-01-03 Syngenta Participations Ag Genes de plantes intervenant dans la defense contre des pathogenes

Non-Patent Citations (1)

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Title
See references of WO2006021558A3 *

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WO2006021558A2 (fr) 2006-03-02

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