WO2001009355A2 - S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION - Google Patents

S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION Download PDF

Info

Publication number
WO2001009355A2
WO2001009355A2 PCT/EP2000/007472 EP0007472W WO0109355A2 WO 2001009355 A2 WO2001009355 A2 WO 2001009355A2 EP 0007472 W EP0007472 W EP 0007472W WO 0109355 A2 WO0109355 A2 WO 0109355A2
Authority
WO
WIPO (PCT)
Prior art keywords
plant
protoporphyrin
nucleotide sequence
methyltransferase
plants
Prior art date
Application number
PCT/EP2000/007472
Other languages
German (de)
English (en)
Other versions
WO2001009355A3 (fr
Inventor
Andreas Reindl
Ralf Reski
Jens Lerchl
Bernhard Grimm
Ali Al-Awadi
Original Assignee
Basf Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Priority to EP00958357A priority Critical patent/EP1198578A2/fr
Priority to AU69908/00A priority patent/AU6990800A/en
Publication of WO2001009355A2 publication Critical patent/WO2001009355A2/fr
Publication of WO2001009355A3 publication Critical patent/WO2001009355A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • Vegetable S-adenosylmethionine Mq-protoporphyrin-lX-O-methyltransferase, plants with an altered chlorophyll content and / or herbicide tolerance and process for their production
  • the invention relates to nucleotide sequences coding for plant S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferases, plants with an altered chlorophyll content, herbicide-tolerant plants and methods for producing such plants.
  • Heme acts as a cofactor in hemoglobin in higher animals, in plants as a cofactor in cytochrome, P450 oxygenases, peroxidases and catalases (for an overview, see Voet and Voet, Biochemie, 1994, VCH; Suzuki et al., 1997, Annual Reviews in Genetics 31 : 61-89; Rüdiger 1997, Phytochemistry 46: 1151-1167) and is therefore an essential component in all aerobic organisms.
  • protoporphyrinogen-IX The last common step of the two biosynthetic pathways is the oxidation of protoporphyrinogen-IX to protoporphyrin-IX, which is catalyzed by the enzyme protoporphyrinogen-IX oxidase (for an overview, see Richter, Biochemie der convinced, 1996, Georg Thieme Verlag).
  • protoporphyrin-IX On the way to chlorophyll, protoporphyrin-IX receives a central Mg through the Mg chelatase, which produces Mg-protoporphyrin-IX.
  • Mg-protoporphyrin-IX-O-methyltransferase (EC 2.1.1.11) leads to Mg-protoporphyrin-13 3 -monomethyl ester, which further in batches characterized steps to chlorophyll a and b is implemented.
  • S-adenosylmethionine Mg-protoporphyrin-IX-O-methyltransferase in chlorophyll biosynthesis has been shown by investigations with inhibitors.
  • Sinefungin a structural analogue of S-adenosylmethionine (SAM), which is secreted by the bacterium Streptomyces, is able to inhibit the S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase, which has been enriched from barley chloroplasts (Vothknecht et al., 1995, Plant Physiology and Biochemistry 33, 759-763). The administration of increasing amounts of sinefungin led to an increasing inhibition of chlorophyll biosynthesis in the barley leaves.
  • PMT is a soluble enzyme in the stroma of plant plastids. Enzyme activities of PMT have already been described in plants, for example in maize (Radmer and Bogorad (1967), Plant Physiol. 42: 463-465), and in photosynthetic bacteria (Gorchein (1972), Biochem, J. 127: 97-106) Service.
  • the Rhodobacter capsulatus bchM gene was identified as a gene sequence encoding a PMT (Bollivar et al. (1994), J. Bacteriol. 176: 5290-5296; Gibson and Hunter (1994) FEBS Letters 352: 127-130).
  • This gene is located in a 46 kB gene cluster, which was initially fully sequenced and contains genes for numerous proteins of bacteriochiorophyll and carotenoid synthesis and the photosynthetic apparatus.
  • the function of the bchM gene product could be demonstrated by interrupting the gene, since after losing the activity of the encoded protein the substrate of the PMT, Mg-protoporphyrin IX, accumulated.
  • the cyanobacterial gene ChIM was found in a cosmid library from Synechocystis 6803 (Jensen et al, 1996, Plant Molecular Biology 30: 1307-1314).
  • the cyanobacterial gene product ChIM has an amino acid identity to the corresponding Rhodobacter protein of 29%.
  • An object of the present invention is thus to provide gene sequences from plants which code for an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase.
  • Another object of the present invention is to provide transgenic plants or parts, tissues or cells thereof with an altered control of the chlorophyll biosynthetic pathway, an altered chlorophyll content and / or a tolerance to herbicides and their use in plant breeding.
  • the present invention relates to a nucleotide sequence coding for a plant S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase or parts, derivatives, homologs or isoforms thereof.
  • a nucleotide sequence encoding a plant S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase is included, which has a sequence according to SEQ ID NO 1 and the plant protein encoded by it an amino acid sequence according to SEQ ID NO 5 contains.
  • this nucleotide sequence codes for an amino acid sequence with the biological activity of an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase from tobacco (Nicotiana tabacum).
  • the present invention also relates to nucleotide sequences coding for a plant S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase, which contain a sequence according to SEQ ID NO 2 or according to SEQ ID NO 3 or according to SEQ ID NO 4.
  • the invention further encompasses nucleotide sequences which code for a protein which contains an amino acid sequence according to SEQ ID NO 6 or SEQ ID NO 7.
  • nucleotide sequences coding for an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase or parts thereof come from Physcomitrella patens.
  • the present invention also includes essentially equivalent, i.e. functionally equivalent nucleotide sequences (allele variations).
  • nucleotide sequences are also selected, selected from the group consisting of a) DNA sequences which comprise a nucleotide sequence which contains the sequence shown in SEQ ID NO. 5 encode specified amino acid sequence or fragments thereof, b) DNA sequences which comprise a nucleotide sequence which the in
  • SEQ ID NO. 6 or SEQ ID NO 7 encode the amino acid sequence indicated or fragments thereof
  • Nucleotide sequence of a), b, c), d) or e) is degenerate, or comprise parts of this nucleotide sequence, g) DNA sequences which are a derivative, analog or fragment of a
  • the present invention also relates to nucleotide sequences which are homologous to one of the aforementioned nucleotide sequences, i. H. has a sequence identity of at least 40%, preferably at least 60%, particularly preferably more than 80% and in particular more than 90% and for a protein with the biological activity of an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase or Parts of it are encoded.
  • the nucleotide sequence coding for an S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase, its parts, derivatives or homologues can be natural, chemically synthesized, modified or artificially generated nucleotide sequences.
  • the nucleotide sequence coding for S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase and its allele variations can be heterologous nucleotide sequences of various others Organisms and mixtures of these and the aforementioned nucleotide sequences contain.
  • both parts of coding nucleotide sequences such as. B. partial cDNAs, as well as so-called full-length clones of a coding structural gene, possibly including regulatory elements.
  • the length of an S-adenosy! Methionine: Mg-protoporphyrin-IX-O-methyltransferase according to the invention can be, for example, in the range from 325 ⁇ 10 amino acid residues to 325 ⁇ 250 amino acid residues, preferably from 325 ⁇ 50 to 325 ⁇ 100 and particularly preferably from 325 ⁇ 25 to 325 ⁇ 50 amino acid residues vary.
  • the “base number” of 325 amino acid residues corresponds to a polypeptide sequence of an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase according to SEQ ID NO 5 encoded by a nucleotide sequence according to SEQ ID NO 1. Consequently, the “base number” of the polypeptide can be in Depending on the nucleotide sequence encoding they also vary.
  • Mg-protoporphyrin-IX-O-methyltransferase means a protein which catalyzes the methylation of protoporphyrin IX.
  • biologically active fragment means that the mediated biological activity is sufficient to influence the chlorophyll content.
  • Mg-protoporphyrinogen-IX-O-methyltransferase are those sequences which, despite a different nucleotide sequence, still have the desired functions (allelic variations).
  • Functional equivalents thus include naturally occurring variants of the sequences described here as well as artificial ones, for example by chemical or Genetic engineering obtained artificial nucleotide sequences adapted to the codon use of a plant. This can be, for example, DNA or RNA molecules, cDNA, genomic DNA, mRNA etc.
  • functionally equivalent sequences include those which have an altered nucleotide sequence which gives the enzyme resistance to inhibitors.
  • a functional equivalent is also understood to mean, in particular, natural or artificial mutations of an originally isolated sequence coding for an S-adenosylmethionine: Mg-protoporphyrinogen-IX-0-methyltransferase, which furthermore show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
  • the present invention also includes those nucleotide sequences which can be obtained by modifying the S-adenosylmethionine: Mg-protoporphyrinogen-IX-0-methyltransferase-
  • the aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also be the insertion of further restriction enzyme interfaces.
  • Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.
  • artificial DNA sequences are suitable as long as they impart the desired properties, as described above.
  • Such artificial DNA sequences can be determined, for example, by back-translation of proteins constructed using molecular modeling, which have S-adenosylmethionine: Mg protoporphyrinogen-IX-0-methyltransferase activity, or by in vitro selection. Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to that for the host plant are particularly suitable specific codon usage.
  • the specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other, known genes of the plant to be transformed.
  • a further variant of the present invention comprises a nucleotide sequence and its allele variations, which codes for a modified S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase or parts, derivatives, homologs or isoforms thereof, which tolerate compounds with herbicidal activity, such as Sinefungin.
  • an in vitro or in vivo (bacterial) mutagenesis is carried out according to the invention, in which correspondingly resistant forms of the gene can be identified by methods known per se. With this technique, for any S-adenosylmethionine: Mg-Protoporphyrinogen-IX-0-
  • Methyltransferase-specific inhibitor-resistant forms of S-adenosylmethionine Mg-protoporphyrinogen-lX-0-methyltransferase can be generated.
  • the present invention thus also relates to a method for isolating a nucleotide sequence coding for a herbicide-tolerant S-adenosylmethionine: Mg-protoporphyrin-IX-0-
  • Methyltransferase wherein a nucleotide sequence according to the invention of the type described above is used in an in vitro or in vivo mutagenesis, then possibly transferred to a single or multicellular host organism, then the organisms which are in the presence of a herbicide ( due to the mutation-modified nucleotide sequence coding for a herbicide-tolerant PMT) can be isolated and finally those in these organisms containing genetically modified nucleotide sequences coding for a herbicide-tolerant S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase can be isolated.
  • S-adenosylmethionine: Mg-protoporphyrinogen-IX-O-methyltransferase gene is characterized in that operatively linked regulatory nucleic acid sequences which control the expression of the coding sequence in a host cell are assigned to it.
  • An operative link is understood to mean the sequential arrangement of, for example, the promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended in the expression of the coding sequence.
  • any promoter that can control the expression of foreign genes in plants is suitable as a promoter.
  • the DNA sequences according to the invention can be expressed in plant cells under the control of constitutive, but also inducible and / or tissue- or development-specific regulatory elements, in particular promoters.
  • constitutive, but also inducible and / or tissue- or development-specific regulatory elements in particular promoters.
  • tissue-specific, for example leaf and / or seed-specific, promoters offers the possibility of determining the chlorophyll content in certain tissue, for example in leaf or. Seed tissue, changing.
  • Other suitable promoters for example, mediate light-induced gene expression in transgenic plants.
  • the promoter can be homologous or heterologous with respect to the plants to be transformed.
  • suitable promoters include the 35S Cauliflower Mosaic Virus RNA promoter and maize ubiquitin promoter for constitutive expression.
  • Suitable seed-specific promoters are, for example, the USP (Bäumlein et al. (1991), Mol. Gen. Genet. 225: 459-467) or the Hordein promoter (Brandt et al. (1985), Carlsberg Res. Commun. 50: 333-345).
  • the promoters mentioned are also particularly suitable for the targeted reduction of the chlorophyll content in transgenic seeds using the DNA sequences according to the invention in connection with the antisense or cosuppression technique. In any case, the person skilled in the art can find suitable promoters in the literature or isolate them from any plants using routine methods.
  • sequences which are preferred but not restricted to the operative linkage are enhancers, transcription terminators, targeting sequences (transit signal sequences) to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in mitochondria, in the endoplasmic reticulum (ER), in the cell nucleus, in Oil bodies or other compartments and translation enhancers such as the 5 'leader sequence from the tobacco mosaic virus (Gallie et al., Nuci. Acids Res. 15 (1987), 8693-871 1). Furthermore, there is a transcription or termination sequence which serves to correctly terminate the transcription and can also be used to add a polyA tail to the transcript, which is assigned a function in stabilizing the transcripts.
  • Such elements are described in the literature (e.g. Gielen, 1989, EMBO J., 8: 23-29) and are interchangeable, e.g. the terminator of the octopine synthase gene from Agrobacterium tumefaciens.
  • the present invention furthermore relates to a gene structure comprising an S-adenosylmethionine: Mg protoporphyrinogen IX-0-methyltransferase gene according to one of the previously described Design variants and operatively linked regulatory nucleotide sequences of the aforementioned type.
  • Chimeric gene constructs are also conceivable here, according to which mixtures of a nucleotide sequence according to the invention fused with homologous or heterologous regulatory structures are to be understood.
  • the gene structure comprises upstream, i.e. at the 5 'end of the coding sequence, a promoter and downstream, i.e. at the 3 'end, a polyadenylation signal and optionally further regulatory elements which are operatively linked to the sequence coding for the S-adenosylmethionine: Mg-protoporphyrinogen-IX-0-methyitransferase gene in between.
  • the present invention also includes a gene structure containing regulatory nucleotide sequences from the group of promoters, enhancers, operators, terminators, polyadenylation signals, targeting sequences, retention signals or translation enhancers. Numerous examples of this are described in the literature. These preferably include those regulatory sequences which are active in the host organisms and / or plants used.
  • any promoter that can control the expression of foreign genes in plants is suitable as promoters of the gene structure.
  • a plant promoter or a plant virus-derived promoter is preferably used.
  • the CAMV 35S promotor from the cauliflower mosaic virus (Franck et al., Cell 21 (1980), 285-294) is particularly preferred.
  • this promoter contains different recognition sequences for transcriptional effectors which, in their entirety, become permanent and constitutive Expression of the introduced gene lead (Benfey et al., EMBO J, 8 (1989), 2195-2202).
  • the gene structure can also contain, for example, a chemically inducible promoter through which the expression of the exogenous S-adenosylmethionine: Mg-protoporphyrinogen-IX-0-methyltransferase gene in the plant can be controlled at a specific point in time.
  • a chemically inducible promoter through which the expression of the exogenous S-adenosylmethionine: Mg-protoporphyrinogen-IX-0-methyltransferase gene in the plant can be controlled at a specific point in time.
  • promoters such. B. the PRP1 promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a salicylic acid-inducible promoter (WO 95119443), a benzene-sulfonamide-inducible (EP-A 388186), a Tetracycline-inducible (Gatz et al., (1992) Plant J.
  • the gene structure can therefore be, for example, a seed-specific promoter (preferably the phaseolin promoter (US 5504200), the USP- (Baumlein, H. et al., Mol. Gen. Genet. (1991) 225 (3), 459-467) or LEB4 promoter (Fiedler and Conrad, 1995), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
  • a seed-specific promoter preferably the phaseolin promoter (US 5504200), the USP- (Baumlein, H. et al., Mol. Gen. Genet. (1991) 225 (3), 459-467) or LEB4 promoter (Fiedler and Conrad, 1995), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
  • a gene construct which contains a constitutive promoter as the regulatory nucleotide sequence. It is equally conceivable for a gene structure according to the invention to contain, as a regulatory nucleotide sequence, a leaf- and / or seed-specific and / or an inducible, preferably a light-inducible promoter.
  • a gene structure is produced by fusing a suitable promoter with a suitable nucleotide sequence encoding an S-adenosylmethionine: Mg-protoporphyrinogen-IX-0-methyltransferase (structural gene) and preferably a DNA inserted between promoter and "structural gene", which codes for a chloroplast-specific transit peptide
  • a suitable promoter with a suitable nucleotide sequence encoding an S-adenosylmethionine: Mg-protoporphyrinogen-IX-0-methyltransferase (structural gene) and preferably a DNA inserted between promoter and "structural gene", which codes for a chloroplast-specific transit peptide
  • the present invention also relates to a gene structure in which the nucleotide sequence coding for a PMT according to the invention of the type described above is not contained in a sense orientation, as in most cases, but in an antisense orientation.
  • Gene structures can also be used whose DNA sequence codes for an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase fusion protein.
  • part of the fusion protein can be a transit peptide which controls the translocation of the polypeptide.
  • Preferred transit peptides are preferred for the chloroplasts, which are cleaved enzymatically after translocation of the S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyitransferase gene into the chloroplasts from the S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase part.
  • the transit peptide which is derived from the plastid S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase or a functional equivalent of this transit peptide is particularly preferred.
  • the S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase fusion proteins according to the invention also contain sequences which are particularly suitable for the improved purification of the S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase, for example via affinity chromatography, and which can then be split off again.
  • a so-called “His tag” or sequences against epitopes of antibodies can be mentioned here as examples.
  • the nucleotide sequence encoding an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase encoding can be synthetically produced or naturally obtained, as well as different from different heterologous S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase gene segments Organisms exist or contain mixtures of synthetic and natural DNA components.
  • nucleotide sequences with codons are generated which are preferred by plants. These codons preferred by plants can be determined from codons with the highest protein frequency, which are expressed in most interesting plant species.
  • various DNA fragments can be changed in such a way that a nucleotide sequence is obtained which is expediently read in the correct direction and equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • the promoter and terminator regions can preferably be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, preferably 1 to 8, particularly preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges.
  • the promoter can be native or homologous as well as foreign or heterologous to the host plant.
  • An embodiment variant of the gene structure according to the invention contains the promoter in the 5'-3 direction of transcription, a DNA sequence which is for an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase gene encodes and a region for transcriptional termination. Different termination areas are interchangeable.
  • Preferred polyadonylation signals are vegetable
  • Polyadenylation signals preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACH ⁇ (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional equivalents.
  • the specific ER retention signal SEKDEL (Schouten, A. et al., Plant Mol. Biol. 30 (1996), 781-792) can also be contained in the gene construct. This triples or quadruples the average level of expression. Other retention signals, which occur naturally in plant and animal proteins located in the ER, can also be used for the structure of the gene structure.
  • the gene construct can be, for example, a constitutive promoter (preferably the CAMV 35 S promoter), the LeB4 signal peptide, which is to be expressed Gen and the ER retention signal may be included.
  • the amino acid sequence KDEL lysine, aspartic acid, glutamic acid, leucine is particularly preferred here as the ER retention signal.
  • the present invention relates to a vector containing, as described above, a nucleotide sequence encoding an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase, to regulatory nucleotide sequences operatively linked thereto, and to additional regulation signals, for example nucleotide sequences for replication in a corresponding one Host cell and / or for integration into the host cell genome.
  • the vector according to the invention can contain a gene structure of the aforementioned type.
  • the gene structures can be cloned into suitable vectors that propagate in host cells such as
  • the invention thus also relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering and which comprise the above-described inventive
  • nucleic acid molecules Contain nucleic acid molecules and if necessary for the transfer of the nucleic acid molecules according to the invention to plants or
  • Plant cells can be used. Suitable vectors are inter alia in "Methods in Plant Molecular Structure"
  • pBR332, pUC series, M13mp series and pACYCI 84 are preferred as cloning vectors. Especially binary vectors are preferred which can replicate both in E. coli and, for example, in agrobacteria.
  • the pBIN19 may be mentioned as an example (Bevan et al., Nucl. Acids Res. 12 (1984), 871 1).
  • the gene structure according to the invention can also be incorporated into the tobacco transformation vector pBIN-AR-TP (FIG. 2).
  • the present invention furthermore comprises a probe for specific hybridization with a DNA sequence or a mRNA derived therefrom coding for an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase or parts thereof, wherein this probe is a coherent sequence of the invention for a contains plant-based PMT coding nucleotide sequence which is at least 10 nucleotides long. This also includes short nucleotide sequences with a length of, for example, 10 to 30, preferably 12 to 15 nucleotides. This includes u. a. also so-called primers.
  • the probe according to the invention is further characterized in that it contains a label suitable for detection, for example a digoxygenin system (Boehringer Mannheim).
  • the invention also relates to a kit for identifying further eukaryotic nucleotide sequences coding for an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase, this kit containing at least one previously described probe and instructions for hybridizing and detecting nucleotide sequences, such as the one described are known from the literature (Boehringer Mannheim or T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratury, Cold Spring Harbor, NY (1989)).
  • This kit is used according to the invention for the isolation of S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase from eukaryotes.
  • the invention also relates to a method for isolating a DNA sequence which codes for a plant S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase, which is characterized in that a cDNA library is created starting from a plant source , this library is used for hybridization with a probe of the type described and the positive cDNA clones are detected and isolated.
  • the present invention relates to an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase or parts, derivatives, homologs or isoforms thereof, which is encoded by one of the previously described embodiment variants of the nucleotide sequences according to the invention or their allelic variations.
  • Isoforms are to be understood as enzymes with the same or comparable substrate and activity specificity, but which have a different primary structure.
  • an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase or parts thereof is included, containing an amino acid sequence according to SEQ ID NO 5 or parts or derivatives or isoforms thereof or mixtures thereof.
  • This is preferably a plant PMT, particularly preferably from tobacco (Nicotiana tabacum).
  • the present invention also relates to an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase or parts thereof containing an amino acid sequence according to SEQ ID NO 6 or SEQ ID NO 7 or parts or derivatives or isoforms thereof or mixtures thereof.
  • This protein or parts thereof preferably originate from Physcomitrella patens.
  • the present invention further relates to a further variation of an S-adenosylmethionine: Mg-protoporphyrin-IX-0- Methyltransferase, which has a tolerance to compounds with herbicidal activity.
  • the present invention further relates to antibodies with the ability to bind specifically to one of the aforementioned S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase.
  • Antibodies are produced according to the invention by injecting a protein or part of a protein coded by an S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase gene according to the invention into animals or animal cells, producing antibodies by immune reaction and the produced antibodies are isolated from the animals or animal cell cultures. According to the invention, these antibodies are used for the specific detection and for the improved purification of S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferases from eukaryotes.
  • the present invention furthermore relates to the transfer of a previously described nucleotide sequence according to the invention and its allelic variations coding for an S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase from plants, for example in the form of the gene construct or vector according to the invention, into suitable host systems.
  • a nucleotide sequence coding for an S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyl transferase from plants is transferred to a suitable host system by genetic engineering methods.
  • Plants can be homologous organisms to this host system.
  • heterologous host systems are also conceivable.
  • it can be microorganisms, such as. B. bacteria, viruses, Act fungi, cyanobacteria, algae or plants, plant tissues or cells.
  • the microorganisms, viruses or fungi are, so to speak, an "intermediate station" before the actual transfer of the nucleotide sequences according to the invention into plant systems.
  • the invention also relates to host cells which, in addition to the nucleic acid molecules according to the invention, contain one or more nucleic acid molecules which are transmitted by genetic engineering or natural means and which carry the genetic information for enzymes involved in chlorophyll biosynthesis.
  • Modified DNA or foreign genes are transferred to a host cell using genetic engineering methods.
  • a wide range of transformation methods has been established here. Suitable methods for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are the protoplast transformation by polyethylene glycol-induced DNA uptake, biolistic methods with the gene gun - the so-called particle bombardment method, the electroporation, the incubation of dry embryos in DNA containing solution, microinjection and Agrobacterium-mediated gene transfer using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti or Ri plasmid, must be connected as a flank region to the genes to be introduced.
  • the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary vector.
  • the intermediate vectors can be integrated by homologous recombination into the Ti or Ri-Plindind of the agrobacteria due to sequences that are homologous to sequences in the T-DNA.
  • Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation).
  • Binary vectors can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria.
  • the agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present.
  • the Agrobacterium transformed in this way is used to transform plant cells.
  • the use of T-DNA for the transformation of plant cells has been intensively investigated and has been sufficiently described in well-known overview articles and manuals for plant transformation.
  • the invention thus relates to a genetically modified single-cell or multicellular host organism and its progeny, comprising, in replicable form, a nucleotide sequence according to the invention and / or a gene structure and / or a vector of the type described above. or multicellular host organism around a microorganism, preferably a bacterium, or a virus or a fungus or a plant or plant cell or plant tissue.
  • nucleic acid molecules according to the invention By providing the nucleic acid molecules according to the invention, it is now possible to use genetic engineering methods to modify plant cells to the extent that they have a new or changed PMT activity compared to genetically unmodified cells, in particular wild-type cells, and as a result thereof there is a change in Chlorophyll biosynthetic performance and / or tolerance to herbicides comes.
  • the invention thus relates to a process for the production of plants or parts, tissues or cells thereof with changed control of chlorophyll biosynthesis, comprising the steps of producing a gene construct and / or a vector of the type described above and transferring the gene construct and / or the vector on plant cells.
  • the procedure for producing plants or parts, tissues or cells thereof with an altered chlorophyll content is analogous, as is the case for a process for Production of herbicide-tolerant plants, parts, tissues or cells thereof.
  • one or more nucleotide sequences according to the invention can be transferred in replicating form into the plant cells.
  • these methods include the regeneration of transgenic plants from the plant cells.
  • the transfer of the nucleotide sequences, gene constructs and / or vectors according to the invention to plant cells includes not only the transfer to isolated cells present in cell culture, but also the transfer to living whole plants, ie to plant cells that are not in cell culture but in intact tissue.
  • the regeneration of the transgenic plants from transgenic plant cells is carried out according to customary regeneration methods using conventional nutrient media and phytohormones.
  • the plants thus obtained can then, if desired, by conventional methods, including molecular biological methods, such as PCR, blot analysis, or biochemical methods for the presence of the introduced DNA, which encodes a protein with the enzymatic activity of a PMT, or for the presence of PMT enzyme activity will be examined.
  • the Detection of the enzymatic activity of PMT can also be determined by a person skilled in the art using protocols available in the literature. Furthermore, one can, for example, lay out the seeds obtained by selfing or crossings on medium which contains a suitable selection agent which matches the selection marker which is transferred together with the PMT-DNA sequence. Based on the germination capacity and the growth of the daughter generation (s) of the seeds and the segregation pattern, conclusions can be drawn about the genotype of the respective plant.
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants can then be regenerated from the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which may contain antibiotics or biocides for the selection of transformed cells.
  • agrobacteria The transformation of plants by agrobacteria is known, among other things, from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol, 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
  • the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker which gives the transformed plant cells resistance to a blocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin and others.
  • the individually selected markers should therefore allow the selection of transformed cells over cells that lack the inserted DNA.
  • Alternative markers are also suitable for this, such as nutritive markers and screening markers (such as GFP, green fluorescent protein).
  • selection markers can also be completely dispensed with, but this is associated with a fairly high need for screening. If the selection marker used is to be removed again after the transformation and identification of successfully transformed cells or plants, various strategies are available to the person skilled in the art. For example, sequence-specific recombinases can be used, for example in the form of retransformation of a starting line expressing recombinase and outcrossing of the recombinase after removal of the selection marker (Reiss et al (1996) Proc. Natl. Acad. Sci. USA 93: 3094-3098; Bayley et al (1992) Plant Mol. Biol. 18: 353-361; Lloyd et al. (1994) Mol. Gen. Genet.
  • the selection marker can also be removed by cotransformation followed by outcrossing.
  • a particularly preferred embodiment of the present invention is a genetically modified plant or parts, tissue or cells thereof and their progeny containing, in replicable form, a nucleotide sequence according to the invention and / or a gene construct and / or a vector of the type described above.
  • the invention further relates to transgenic plant cells or plants comprising transgenic plant cells or parts and products thereof in which the nucleic acid molecules according to the invention are present as integrated in the plant genome.
  • the invention also relates to plants in whose cells the nucleic acid sequence according to the invention is present in self-replicating form, ie the Plant cell contains the foreign DNA on an independent nucleic acid molecule, e.g. B. a plasmid. This is known as transient expression.
  • the genetically modified plant or its parts, tissues or cells and their progeny are characterized in that, due to the presence and expression of the nucleotide sequences according to the invention, they synthesize proteins which the corresponding non-genetically modified plant, e.g. B. the wild type, not produced.
  • the invention encompasses a genetically modified plant or parts, tissues or cells thereof and their progeny which has a net productivity of S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase which is greater than that of the correspondingly non-genetically modified plant, in particular one Wild type, is elevated.
  • this is preferably a genetically modified plant or parts, tissue or cells thereof and their progeny which has a modified chlorophyll content which is increased or decreased compared to that of the correspondingly non-genetically modified plant.
  • the present invention also relates to a genetically modified plant or parts, tissue or cells thereof and their progeny which has an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase which has a tolerance to compounds having a herbicidal action.
  • the present invention thus also relates to a herbicide-tolerant plant, plant tissue, plant cell and its progeny, containing in replicating form a previously described S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase gene, a gene structure or a vector containing one Gene structure of the previous
  • S-adenosylmethionine Mg-protoporphyrin-IX-0-methyltransferase gene, a gene structure or a vector containing one Gene structure of the previous
  • the type mentioned is also the subject of the invention of herbicide-tolerant plants, plant tissues, plant cells and their progeny which have a net productivity of S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase, which is increased compared to the correspondingly non-genetically modified form
  • the invention further relates to a transgenic plant and its progeny which expresses an S-aden
  • a particularly preferred embodiment variant relates to a genetically modified plant or parts thereof, plant tissue, cells and their progeny, which has an altered control of chlorophyll biosynthesis and / or an altered chlorophyll content and / or a herbicide tolerance in relation to a corresponding non-genetically modified plant.
  • the invention also relates to those plants in which the transfer of the nucleotide sequences according to the invention leads to a reduction in the chlorophyll content.
  • Such reduced chlorophyll biosynthesis performance can be achieved, for example, by the transfer of antisense constructs or by others
  • Suppression mechanisms such as cosuppression, can be achieved.
  • these plants are characterized by reduced growth, a bleached phenotype, reduced RNA and protein contents for PMT and a greatly reduced chlorophyll content.
  • the use of such transgenic plants in the elucidation of key enzymes and key metabolites of the chlorophyll biosynthetic pathway or for the identification or (new) synthesis of herbicides or compounds with an inhibitory effect on these central closing enzymes of the chlorophyll biosynthetic pathway is also included according to the invention.
  • any plant can be used for a transfer of the genetic material according to the invention.
  • These can be monocot or dicot crops.
  • monocotyledonous plants are plants belonging to the genera Avena (oats), Triticum (wheat), Seeale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (Corn) belong.
  • dicotyledonous crops are to name legumes, such as legumes and in particular alfalfa, soybean, rapeseed, tomato, sugar beet, canola, potatoes, ornamental plants or trees.
  • Other useful plants can be, for example, fruit (in particular apples, pears, cherries, grapes, citrus, pineapple and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal, cotton, flax, sunflower and medicinal plants and pasture grasses and forage plants.
  • the cereals wheat, rye, oats, barley, rice, corn and millet, feed grain, sugar beet, rapeseed, soybean, tomato, potato, sweet grasses, feed grasses and clover are particularly preferred.
  • the invention relates in particular to conventional food or fodder plants.
  • peanut, lentil, broad bean, black beet, buckwheat, carrot, sunflower, Jerusalem artichoke, turnip, white mustard, turnip and stubble are worth mentioning.
  • the invention also relates to seeds of the genetically modified plants according to the invention.
  • the invention also relates to reproductive material from a single or multicellular host organism or from transgenic plants, plant tissues or cells of the type described above.
  • reproductive material or crop products are those according to the invention to understand genetically modified plants, for example seeds, fruits, cuttings, tubers, rhizomes, etc., and parts of these plants, such as protoplasts, plant cells and calli.
  • the present invention also includes a method for identifying cells from a cell population that contain a herbicide-tolerant S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase.
  • This method comprises the following steps: mutagenesis of the cell population, by chemical or genetic engineering treatment of the cells, cultivation of the population of cells in the presence of at least one compound with herbicidal activity which inhibits the activity of S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase inhibits selection of cells whose growth is unchanged, ie is not inhibited.
  • process steps such as regeneration of genetically modified plants from the herbicide-tolerant cells and isolation, identification and / or characterization of a herbicide-tolerant S-adenosylmethionine: Mg-protoporphyrin-IX-O-methyltransferase from the selected ones Cells or, if necessary, isolation, identification and / or characterization of a nucleotide sequence coding for a herbicide-tolerant S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase from the selected cells.
  • the present invention also relates to a method and a measuring system for identifying compounds having a herbicidal action.
  • This measuring system contains at least one S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase as well
  • the S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase encoded by a corresponding nucleotide sequence is purified in preceding steps, then this S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase with compounds which, for example, are catalytic or the regulatory center of the enzyme, incubated and then the specific activity of S-adenosylmethionine: Mg-protoporphyrin-IX-0-
  • Methyltransferase measured is an in vivo measuring system, such as an organism transformed with a nucleotide sequence coding for an S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase, to which potentially herbicidal compounds are added. Due to the measurement of differences in specific enzyme activity, i.e. a reduction in the specific PMT activity, the effect of the added compounds can be identified as a herbicide or inhibitor of PMT.
  • the present invention further relates to the use of a nucleotide sequence according to the invention of the type described and / or a probe created or isolated on the basis of the nucleotide sequence according to the invention for isolating and / or amplifying a nucleotide sequence, preferably from eukaryotes which are suitable for an S- Adenosyimethionine: Mg-protoporphyrin-IX-0-methyltransferase or parts thereof.
  • the present invention comprises the use of a nucleotide sequence according to the invention for the production of plants or parts, tissues or cells thereof with an altered control of the chlorophyll biosynthesis and / or an altered chlorophyll content and / or an altered herbicide tolerance.
  • Herbicide-tolerant forms of this enzyme Compared to chemical syntheses, the use of these enzymes for the biological (in vitro) production of chlorophyll or its derivatives as an economically interesting plant vitamin is conceivable.
  • the invention also relates to the use of a concentrated S-adenosylmethionine: Mg-
  • DNA sequences and the proteins encoded by them represent an extremely valuable “target” for herbicide research.
  • the proteins according to the invention with enzymatic PMT activity can be used for X-ray structure analysis, NMR spectroscopy, molecular modeiing and drug design are used to identify or synthesize inhibitors and / or effectors of PMT and thus potential herbicides based on the knowledge gained from these methods.
  • the present invention also provides for the use of the plants described above and genetically modified according to the invention in the areas of plant breeding, plant protection and agriculture.
  • Cloning methods such as Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA were as with Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press, ISBN 0-87969-309-6).
  • the transformation of Agrobacterium tumefaciens was carried out according to the method of Höfgen and Wiilmitzer (Nucl. Acid Res. (1988) 16, 9877).
  • the agrobacteria were grown in YEB medium (Vervliet et al., J. Gen. Virol. (1 975) 26, 33),
  • the bacterial strains used (E. coli, XL-1 Blue) were from
  • Agrobacterium strain Agrobacterium tumefaciens, C5SC1 with the plasmid pGV2260 or pGV3B50kan
  • the one used for plant transformation Agrobacterium strain was developed by Deblaere et al. in nucl. Acids Res. 13 (1985), 4777.
  • the LBA4404 agrobacterial strain (Clontech) or other suitable strains can be used.
  • the vectors pUC19 (Yanish-Perron, Gene 33 (1985), 103-119) pBluescript SK (Stratagene, Heidelberg), PGEM-T (Promega), pZerO (Invitrogen), pBIN19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711-8720) and pBINAR (Höfgen and Willmitzer, Plant Science 66 (1990), 221-230).
  • RNA from 9-day-old Protonema was prepared according to one of Logemann et al. (Anal. Biochem. (1987) 163,21).
  • the poly (A) RNA was then purified via oligo (dT) cellulose type 7 (Pharmacia, Freiburg) according to the manufacturer's instructions. After the photometric concentration had been determined, 5 ⁇ g of the RNA thus obtained were used for the cDNA synthesis. All chemicals and enzymes required for the production of the cDNA were obtained from Stratagene (La Jolla CA 9203-7, USA). The methods used were carried out according to the manufacturer's instructions.
  • the synthesis of the first and second strand of the cDNA was carried out using the Lambda ZAPII-CDNA synthesis kit.
  • the double-stranded cDNAs obtained were then provided with EcoRI adapters and cloned into an EcoRI-cleaved lambda ZAPII vector.
  • EcoRI-cleaved lambda ZAPII vector After in vitro packaging (Gigapack II packaging extract) of the recombinant lambda DNA, XL-1 E. coli cells (Stratagene, Heidelberg) were transformed. The titer of the cDNA library was determined by counting the plaques formed.
  • so-called EST sequences are produced (Expressed Sequence Tags) procedure. According to known methods, such as. B. by homology comparisons with sequences stored in databases, potential candidates of EST sequences which code for a (partial) PMT can be narrowed down and / or identified from a cDNA bank.
  • SEQ ID NO 2 A partial DNA sequence of the S-adenosylmethionine: Mg-Protoporphy ⁇ in-IX-O-methyltransferase gene from Physcomitrella patens is shown in SEQ ID NO 2 or 3 or 4, the sequence according to SEQ ID NO 2 being the 5 'end of the gene coding for S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase and SEQ ID NO 3 represents the 3 'end of this gene from Physcomitrella patens. Both fragments are separated from each other by only about 4 amino acid residues.
  • the fragments according to the invention according to SEQ ID NO 2, 3 and 4 can thus be combined in a simple manner by the person skilled in the art into a gene of entire length.
  • a nucleotide sequence can be isolated as a full-length clone of eukaryotic PMT genes, as described above.
  • the partial amino acid sequence of S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase corresponding to SEQ ID NO 2 or SEQ ID NO 3 is shown in SEQ ID NO 6 or SEQ ID NO 7.
  • SEQ ID NO 2 which was radioactively labeled using a "Multiprime DNA labeling System” (Amersham Buchler) in the presence of ⁇ - 32 P-dCTP (specific activity 3000 Ci / mmol) according to the manufacturer's instructions.
  • the membrane was hybridized after prehybridization at 42 ° C. in PEG buffer (Amasino (1986) Anal. Biochem. 152, 304-307) for 12-16 hours. The filters were then washed 3 ⁇ 20 minutes in 2 ⁇ SSC, 0.1% SDS at 42 ° C. Positive hybridizing phages were visualized by autoradiography and purified by standard techniques.
  • S-adenosylmethionine Mg-protoporphyrin-IX-O-methyltransferase gene in expression vectors of heterologous expression systems and detection of the enzymatic activity.
  • a clone coding for a full length S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase sequence was identified using the partial DNA sequence of the S-adenosylmethionine: Mg-protoporphyrin-IX- 0-Methyltransferase identified in SEQ ID NO 2.
  • Expression vectors are suitable for the expression of recombinant proteins in E. coli, but also baculovirus vectors for the expression of S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase in
  • Insect cells Bacterial expression vectors are derived, for example, from pBR322 and carry a bacteriophage T7 promoter for expression.
  • the plasmid is propagated in an E. coli strain which carries an inducible gene for the T7 polymerase (for example JM109 (DE3); Promega).
  • the expression of the recombinant protein is activated via the induction of the T7 polymerase by IPTG.
  • IPTG-inducible systems from Qiagen are available (pQE vectors) or Novagen (pET vectors). Depending on the interfaces available, there are all vectors with all reading frames.
  • the one with the S-adenosylmethionine Mg-protoporphyrin-IX-0-
  • Vectors containing methyltransferase-transformed E. coli strain was incubated in the culture medium "2xYT" (per 1 liter: bacto-trypton 16 g, yeast extract 10 g, NaCl 1 5 g). The cultivation took place at 37 ° C up to an OD 5 eo of 0.6. After the addition of IPTG (1 mM), the growth continued for a further 10 min at 37 ° C., then for a further 4 h at 28 ° C. until the harvest. The cells were centrifuged off and washed in 1% NaCl.
  • Plant transformation vector pBIN19AR-TP pBIN19AR-TP contains a plastid targeting sequence (TP; see FIG. 2) integrated in the polylinker of the vector, which was inserted via the interfaces Kpnl (as Asp718 isoschizomer) and BamHI (Höfgen and Willmitzer, Plant Science 66 (1990 ), 221-230).
  • TP plastid targeting sequence
  • the 555 base pair fragment coding for the Physcomitrella-S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase according to SEQ ID NO 2 was converted into a binary vector (PBIN-AR-TP; see FIG. 2B) in antisense orientation cloned under control of the 35S promoter.
  • the following primers were chosen for cloning the S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase into a binary vector in an antisense orientation.
  • PpS adenosylmethionine Mg protoporphyrin IX-0-methyltransferase ASpBINI forward 5 'TATGTCGACTATCGAATTCGGCACGAGCT
  • PpS adenosylmethionine Mg protoporphyrin IX-0-methyltransferase ASpBIN2 backwards 5 'TATGGATCCAGCTTTGGAAACGGGAACGCA 3'
  • the PCR product was purified using the Gene Clean Kit (Dianova GmbH, Hilden) and digested with Sall and BamHI.
  • the vector pBIN19AR-TP09 was also cut with Sall and BamHI, which additionally contains the transit peptide of the transketolase from potato behind the CAMV 35S promoter.
  • the heterologous signal sequence (from potato) serves as an additionally detectable sequence in transgenic tobacco plants (via Northern blot or via PCR), beyond the detection of S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase antisense RNA.
  • the construct is shown in Fig. 2B.
  • This construct was transformed into tobacco by Agrobacterium-mediated transformation. Regenerated plants were examined for S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase mRNA amounts. All examined antisense plants with reduced S-adenosyimethionine: Mg-protoporphyrin-IX-0- Amounts of methyl transferase mRNA showed significant differences in terms of plant size. Plants were found that were reduced in size and plants that showed significant bleaching. These bleaches are due to greatly reduced amounts of chlorophyll. The chlorophyll amounts were determined as described in Lichtenthaler and Wellbum (1983) with 100% acetone extracts.
  • the reaction takes place in a total volume of 150 ⁇ l with 20 ⁇ g protein extract (heterologously expressed as His-Tag protein in E. coli with or without subsequent affinity purification via NTA agarose), 0.35 M S-adenosylmethionine (SAM; Sigma) or 3.7 kBq [Methyl-C] SAM (NEN, Boston, USA; specific activity 2.1 GBq mmol "1 ), 20 ⁇ M Mg-protoporphyrin-IX, 0.1 M tricine-NaOH (pH 7.9), 0.3 M glycerol, 25 mM MgCl 2 and 1 mM dithiotreitol.
  • SAM S-adenosylmethionine
  • Homocysteine has free sufhydryl groups that react with the Ellman reagent (5,5'-dithio-bis (2-nitrobenzoic acid; DTNB; Sigma). The detection is carried out photometrically at an absorption wavelength of 412 nm.
  • DTNB is solved according to the manufacturer's instructions (3.96 mg / ml in 0.1 M phosphate buffer pH 7.0), and in a volume ratio of 1: 500 to the batch to be measured (20 ⁇ g E. coli total protein extract in 0.1 M phosphate powder pH 8.0) . The incubation was repeated several times during the up to 45 min Absorbance at 412 nm determined according to the following formula:
  • tobacco leaf disks with sequences of S-adenosylmethionine: Mg-protoporphyrin-IX-0- Transformed methyl transferase from P. patens To transform tobacco plants, 10 ml of an overnight culture of Agrobacterium tumefaciens grown under selection were centrifuged off, the supernatant was discarded and the bacteria were resuspended in the same volume of antibiotic-free medium. Leaf disks of sterile plants (diameter approx. 1 cm) were bathed in this bacterial suspension in a sterile petri dish.
  • the leaf disks were then placed in Petri dishes on MS medium (Murashige and Skoog, Physial, Plant (1962) 15, 473) with 2% sucrose and 0.8% Bacto agar. After 2 days of incubation in the dark at 25 ° C, they were on MS medium with 100 mg / l kanamycin, 500 mg / l claforan, 1 mg / l benzylaminopurine (BAP), 0.2 mg / l naphthylacetic acid (NAA), 1.6% glucose and 0.8% Bacto agar transferred and cultivation continued (16 hours light / 8 hours dark). Growing shoots were transferred to hormone-free MS medium with 2% sucrose, 250 mg / l Claforan and 0.8% Bacto-Agar.
  • the cDNA sequence coding for the PMT from tobacco was determined by screening a lambda ZAP II cDNA library from Nicotiana tabacum (SR1, Stratagene, USA) using a partial EST clone from the moss Physcomitrella ( SEQ ID NO 4) identified according to a standardized protocol.
  • a 548 bp restriction fragment of the EST sequence according to SEQ ID NO 4 was used as a probe for the hybridization of the cDNA library and radioactively labeled with cc-32-P-DCTP using the random primer labeling kit (GIBCO Life Technologies, Eggenstein).
  • the hybridization was carried out according to the following protocol: a) 4 hours prehybridization at 51 ° C with a hybridization solution of the following composition: 5 x SSC, 0.1% SDS, 5 x Denhardts reagent, 100 ⁇ g / ml denatured fish DNA, b) 16 hours main hybridization at 51 ° C with fresh hybridization solution of the following composition: 5 x SSC, 0.1% SDS, 5 x Denhardts reagent, 100 ⁇ g / ml denatured fish DNA with radioactively labeled cDNA probe, c) washing conditions: 1x with 5xSSC, 0.1% SDS , 10 minutes, 51 ° C; 2x with 2xSSC, 0.1% SDS, 10 minutes each, 51 ° C.
  • the PMT cDNA sequence identified and shown in SEQ ID NO 1 comprises 1134 base pairs (without the polyA tail) with a start codon at position 12-14 and a stop codon at position 987-989. Nucleotides 12-986 encode 325 amino acids.
  • the deduced amino acid sequence of the PMT gene is shown in SEQ ID NO 5.
  • the open reading frame for the complete unprocessed protein using the oligonucleotide primers was forward primers: Exe-F 1 (Ncol) 5'-GAT CCC ATG GCT TTC TCT TCG CCA CTA TTC-3 'and reverse primer:
  • Exe-RI 5'-GAT CGG ATC CGC AGG GAC AGC CTC AAT AAG CTT-3 'from the DNA sequence according to SEQ ID NO: 1 amplified by PCR.
  • the PCR conditions were as follows: 5 min. Denaturation at 95 ° C, then for 25 cycles: 1 min. At 94 ° C, 12 min. At 60 ° C / 1 min. At 72 ° C, finally another 3 min Extension at 72 ° C.
  • the PCR was carried out with the PWO enzyme from Stratagene (La Jolla, USA) on a ThermoCycler 9700 from Perkin-Elmer.
  • the amplified PCR fragment was purified using the Qiagen PCR Purification Kit (Qiagen, Hilden), cut with the restriction enzymes Ncol and BamHI (Amersharn, Freiburg, Germany) and ligated into the expression vector pET20B (Novagen) cut with the same enzymes (ligation with TF-DNA ligase from Amersharn for 20 hours at 15 ° C).
  • This pET vector expresses the foreign protein under the control of a promoter recognized by the T7 polymerase and encodes the PelB leader sequence at the N-terminal end of the recombinant protein and a histidine tag at the C-terminal end (FW Studier et al. (1990 ), Meth. Enzymol. 185: 60-89).
  • the synthesis of the foreign protein is made possible after the induction of the synthesis of the bacteriophage T7 RNA polymerase in the E. coli cells.
  • the ligated vector was transformed into the E. coli strain MOS blue (Amersharn, Freiburg) and then into the strain E coli BL 21 (Novagen, Madison, Wl, USA).
  • the E. coli strain BL21 was grown in LB medium to an optical density of 0.6, and then the expression was increased by adding 1 mM IPTG for 5 Hours induced. A recombinant protein with a molecular weight of 33 kDa was synthesized. The protein was in the soluble protein fraction of disrupted E. coli cells.
  • the overexpressed protein was purified using the TALON beads (Clontech) on a cobalt affinity column according to the manufacturer's instructions.
  • the purified protein was infected in rabbits for antibody production.
  • the enzymatic detection of the recombinant protein was carried out according to the method of Gibson and Hunter (FEBS Letters 352: 127-130).
  • the bacterial extract was in a 500 ul assay volume in a buffer consisting of 50 mM Tris-HCl, pH 8.4, 5 MM EGTA with 20 gM Mg protoporphyrin IX (Porphyrin Products Inc., Utah, USA) and 0.5 MM S-Adenosyl-L-Methioni ⁇ (Sigrna, Deisenhofen) incubated as a cofactor at 33 ° C for 60 minutes. At six measuring points (0 sec., 30 sec., 10 min., 25 min., 40 min.
  • Mg porphyrins Diluted 8-fold for the analysis of the Mg porphyrins on a HPLC system from Waters using a Nova-Pak C 1.
  • the Mg porphyrins were eluted with the following linear gradient: from solution A (10% 1 M NH 4 acetate, 10% methanol) to solution B (10% 1 M NH acetate, 90% methanol) within the first 7 minutes , from the 7th to the 21st minute 100% solution B, from the 21st to the 23rd minute of solution B to solution A, from the 23rd to the 29th minute solution A.
  • the elution profile of the Mg porphyrins from the extracts of the PMT assay, which were removed at the indicated reaction times, are shown in FIG. 3.
  • Mg protoporphyrin usually has an elution time of 14.8 minutes and Mg protoporphyrin IX monomethyl ester has an elution time of 16.25 minutes.
  • the DNA sequence according to SEQ ID NO 1 was carried out using the restriction enzymes Smal and Sall in the multiple cloning interface of the pBluescript vector (Stratagene, Amsterdam, Netherlands) cut out of the vector and in sense orientation into the binary vector BinAR-TX (Höfgen and Willmitzer (1 990) Plant Science 66: 221-23 0), a pBIB descendant, which was digested with the same restriction endonucleases, ligated in behind the CAMV 35S promoter.
  • a restriction map of the vector BinAR-TX is attached as Fig. 4.
  • the recombinant vector pPMTbin was then transformed into Agrobacterium tumefaciens (Stanun GV2260; Horsch et al. (1,985), Science 227, 1229-123 1) and for the transformation of tobacco plants (SNN) by means of the leaf disc transformation technique (Horsch et al., supra) used.
  • an overnight culture of the corresponding Agrobacterium tumefaciens clone was centrifuged for 10 minutes at 5000 rpm, and the bacteria were resuspended in 2YT medium.
  • Young tobacco leaves one Sterile culture (Nicotiana tabacum cv. Samsun NN) was cut into small pieces of about 1 cm 2 in size and placed briefly in the bacterial suspension. The leaf pieces were then placed on MS medium (Murashige and Skoog (1 962), Physiol. Plant. 15, 473; 0.7% agar) and incubated for two days in the dark.
  • the leaf pieces were then sprouted on MS medium (0.7% agar) with 1.6% glucose, 1 mg / l 6-benzylaminopurine, 0.2 mg / l naphthylacetic acid, 500 mg / l claforan (Cefotaxim, Hoechst, Frankfurt) and 50 mg / l kanamycin.
  • the medium was changed every seven to ten days.
  • shoots had developed the leaf pieces were transferred to glass jars containing the same medium. Resulting shoots were cut off and placed on MS medium with 2% sucrose and 250 mg / l Claforan and regenerated to whole plants.
  • Example 11 in which transgenic plants are described which, because of the overexpression of the DNA sequence according to the invention, have an increased activity of the PMT, the cDNA sequence was used for the production of transgenic tobacco plants which have a reduced activity of PMT SEQ ID NO 1 was cut out of the pBluescript vector using the restriction enzymes EcoRV and Xbal and ligated into a binary vector BinAR-TX digested with the same enzymes behind the 35S promoter of CAMV (see FIG. 4). The recombinant plasmid was first transferred into the E. coli strain pMOS and then into the Agrobacterium tumefaciens strain GV 2260.
  • the recombinant agrobacteria with the binary vector pPMTASdin were integrated into the tobacco genome according to the leaf disk transformation (see Example 11).
  • the transformants with the PMT sense gene construct showed two different external appearances. About 30 plants showed pale green to green-yellowish leaves and reduced growth. The remaining lines showed no noticeable differences compared to the control plants, although the presence of the transgene was also proven in these lines by means of genomic Southern blot analysis.
  • the PMT antisense plants showed a reduced growth compared to the control plants, a bleached phenotype, reduced RNA and protein contents for PMT and a greatly reduced chlorophyll content.
  • the primary transformants showed one to several copies of the PMT transgene compared to the control plants.
  • Three antisense RNA synthesizing lines were subjected to detailed analysis.
  • the transgenic plants with the copies of the PMT transgene in sense or antisense orientation were analyzed in Southern blot methods.
  • the hybridization with one labeled cDNA fragment for PMT resulted in one to several additional hybridization bands of the genomic DNA digested with a restriction enzyme.
  • a Northem blot analysis showed an increased amount of specific RNA compared to the PMT-RNA contents of the control plants in the case of the PMT sense lines and a reduced amount in the case of the transformants with PMT antisense genes.
  • Increased PMT contents could be determined by means of Western blot analysis in the lines with PMT sense gene constructs and reduced PMT contents in lines with the PMTA antisense construct.
  • the PMT activity of the transformants was determined in vivo. In parallel to the increased protein contents, an increased enzyme activity could be determined for the PMT sense lines, while the lines with antisense RNA showed reduced activities (FIG. 6).
  • ALA synthesis is the rate-limiting step of tetrapyrrole synthesis and is influenced by many external factors (such as light, day / night rhythm, light intensity) and endogenous factors (such as endogenous clock, hormones, development). The results imply a regulatory mechanism that aligns the activities of the early steps with those of the later steps, here that of the PMT.
  • the adaptation of the ALA biosynthesis performance to the activities in the Mg porphyrin synthesis pathway is believed to be based on a joint coordination of the transcriptional and post-translational expression control.
  • Fig. 1 Illustration of the three cassettes for the three different reading frames of the plastid transit peptide of the plastid S-adenosylmethionine: Mg-protoporphyrin-IX-0-methyltransferase from potato.
  • Fig. 2 (A): Plant transformation vector pBIN19AR-TP.
  • TK-Tp transit peptide of the transketolase from potato
  • OCS octopine synthase terminator
  • Fig. 3 Enzyme activity of the recombinant Mg-PMT protein shown as a gene map and elution profile of the Mg porphyrins from the extracts of the PMT enzyme test.
  • Fig. 5 Bar chart showing the Mg-porphyrin content of selected transgenic Mg-PMT tobacco plants containing Gene constructs with coding nucleotide sequences in sense and
  • Antisense orientation referred to as sense and antisense.
  • Fig. 6 Bar chart to show the Mg-PMT activity in modified tobacco plants (Mg-PMT content after 1 hour of incubation).
  • Fig. 7 Bar chart to show the ALA content in transgenic Mg-PMT tobacco lines containing gene constructs with coding nucleotide sequences in sense and antisense orientation, referred to as sense and antisense.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Nutrition Science (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

La présente invention concerne une séquence nucléotidique codant pour une S-adénosylméthionin:Mg-protoporphyrin-IX-O-méthyltransférase végétale, un procédé de production de végétaux génétiquement modifiés sur lesquels peuvent s'effectuer différents contrôles du mode de biosynthèse de la chlorophylle et/ou ayant une teneur en chlorophylle variable et/ou ayant une tolérance aux herbicides variable. L'invention a pour objet des végétaux à teneur en chlorophylle variable ainsi que des végétaux présentant une tolérance aux herbicides. Cette invention concerne également l'utilisation des séquences nucléotidiques PMT végétales de l'invention pour modifier la teneur en chlorophylle de végétaux transgéniques ainsi que pour la détection d'effecteurs, en particulier d'inhibiteurs de PMT végétale ainsi que des composés à action herbicide.
PCT/EP2000/007472 1999-08-03 2000-08-02 S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION WO2001009355A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP00958357A EP1198578A2 (fr) 1999-08-03 2000-08-02 S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION
AU69908/00A AU6990800A (en) 1999-08-03 2000-08-02 Plant

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19935610.6 1999-08-03
DE19935610 1999-08-03
DE10014114.5 2000-03-22
DE10014114 2000-03-22

Publications (2)

Publication Number Publication Date
WO2001009355A2 true WO2001009355A2 (fr) 2001-02-08
WO2001009355A3 WO2001009355A3 (fr) 2001-09-07

Family

ID=26004952

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/007472 WO2001009355A2 (fr) 1999-08-03 2000-08-02 S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION

Country Status (3)

Country Link
EP (1) EP1198578A2 (fr)
AU (1) AU6990800A (fr)
WO (1) WO2001009355A2 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998024920A2 (fr) * 1996-12-04 1998-06-11 Institut für Pflanzengenetik und Kulturpflanzenforschung Comment influer sur la biosynthese de la chlorophylle chez les vegetaux
WO1998049330A1 (fr) * 1997-04-25 1998-11-05 Hoechst Schering Agrevo Gmbh Sequences d'adn codant pour la sous-unite chld de chelatases vegetales de magnesium pour determiner leurs effets
WO1999022011A1 (fr) * 1997-10-29 1999-05-06 Institut für Pflanzengenetik und Kulturpflanzenforschung Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses
EP1033405A2 (fr) * 1999-02-25 2000-09-06 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998024920A2 (fr) * 1996-12-04 1998-06-11 Institut für Pflanzengenetik und Kulturpflanzenforschung Comment influer sur la biosynthese de la chlorophylle chez les vegetaux
WO1998049330A1 (fr) * 1997-04-25 1998-11-05 Hoechst Schering Agrevo Gmbh Sequences d'adn codant pour la sous-unite chld de chelatases vegetales de magnesium pour determiner leurs effets
WO1999022011A1 (fr) * 1997-10-29 1999-05-06 Institut für Pflanzengenetik und Kulturpflanzenforschung Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses
EP1033405A2 (fr) * 1999-02-25 2000-09-06 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1976 ELLSWORTH R K ET AL: "BIOSYNTHESIS AND INHIBITION OF LEVO S ADENOSYL-L METHIONINE MAGNESIUM PROTO PORPHYRIN METHYL TRANSFERASE OF WHEAT" Database accession no. PREV197763047345 XP002164152 & PHOTOSYNTHETICA (PRAGUE), Bd. 10, Nr. 3, 1976, Seiten 291-301, ISSN: 0300-3604 *
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1978 SHIEH J ET AL: "PROPERTIES OF S ADENOSYL-L METHIONINE MAGNESIUM PROTO PORPHYRIN IX METHYL TRANSFERASE EC-2.1.1.11 FROM BARLEY" Database accession no. PREV197967024379 XP002164155 & PLANT AND CELL PHYSIOLOGY, Bd. 19, Nr. 6, 1978, Seiten 1051-1060, EN ISSN: 0032-0781 *
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1995 VOTHKNECHT UTE C ET AL: "Sinefungin inhibits chlorophyll synthesis by blocking the S-adenosyl-methionine: Mg-protoporphyrin IX O-methyltransferase in greening barley leaves." Database accession no. PREV199698639700 XP002164150 & PLANT PHYSIOLOGY AND BIOCHEMISTRY (MONTROUGE), Bd. 33, Nr. 6, 1995, Seiten 759-763, ISSN: 0981-9428 *
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1996 AVERINA N G ET AL: "Formation of chlorophyll biosynthesis intermediates in bean, cucumber and barley seedlings treated with 5-aminolevulinic acid and homocysteine." Database accession no. PREV199698736621 XP002164151 & PHOTOSYNTHETICA (PRAGUE), Bd. 32, Nr. 1, 1996, Seiten 45-52, ISSN: 0300-3604 *
DATABASE EMBL [Online] ACCESSION NO: AF213968, 17. Mai 2000 (2000-05-17) ALAWADY A.E., ET AL.: "Nicotiana tabacum S-adenosyl-L-methionine Mg-protoporphyrin IX methyltranserase (Chl M) mRNA, complete cds; chloroplast gene for chloroplast product." XP002164154 *
DATABASE EMBL [Online] ACCESSION NO: AI774803, 30. Juni 1999 (1999-06-30) D' ASCENZO M., ET AL.: "EST255903 tomato resistant, Cornell Lycopersicon esculentum cDNA clone cLER13K22, mRNA sequence." XP002164149 *
DATABASE EMBL [Online] ACCESSION NO: AI777135, 30. Juni 1999 (1999-06-30) D' ASCENZO M., ET AL.: "EST258100 tomato resistant, Cornell Lycopersicon esculentum cDNA clone cLER20O2, mRNA sequence." XP002164148 *
DATABASE EMBL [Online] ACCESSION NO: AI861012, 22. Juli 1999 (1999-07-22) CUSHMAN J.C.;: "L30-1226T3 Ice plant Lambda Uni-Zap XR expression library, 30 hours NaCl treatment Mesembryanthemum crystallinum cDNA clone L30-1226 5' similar to Magnesium-protoporphyrin IX methyltransferase (AL035523 )[Arabidopsis thaliana], mRNA sequence." XP002164147 *
DATABASE EMBL [Online] ACCESSION NO: AL035396, 10. Februar 1999 (1999-02-10) BEVAN M.,: "Arabidopsis thaliana DNA chromosome 4, BAC clone F24A6 (ESSAII project)" XP002164145 in der Anmeldung erwähnt *
DATABASE EMBL [Online] ACCESSION NO: AL035523, 24. Februar 1999 (1999-02-24) BEVAN M., ET AL.: "Arabidopsis thaliana DNA chromosome 4, BAC clone F13M23 (ESSAII project)" XP002164146 in der Anmeldung erwähnt *
DATABASE EMBL [Online] ACCESSION NO: AW038070, 17. September 1999 (1999-09-17) D'ASCENZO M., ET AL.: "EST279727 tomato mixed elicitor, BTI Lycopersicon esculentum cDNA clone cLET2P9, mRNA sequence." XP002164153 *
SMITH CRAIG A ET AL: "Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus." PLANT MOLECULAR BIOLOGY, Bd. 30, Nr. 6, 1996, Seiten 1307-1314, XP002164144 ISSN: 0167-4412 & DATABASE EMBL [Online] ACCESSION NO: L47126, 24. Januar 1996 (1996-01-24) *

Also Published As

Publication number Publication date
WO2001009355A3 (fr) 2001-09-07
AU6990800A (en) 2001-02-19
EP1198578A2 (fr) 2002-04-24

Similar Documents

Publication Publication Date Title
WO1999004021A1 (fr) Sequence d'adn codant pour une hydroxyphenylpyruvate dioxygenase et sa surproduction dans des plantes
WO2001014569A2 (fr) Augmentation de la teneur en polysaccharides dans des plantes
WO2000008169A1 (fr) Sequence adn codant pour une 1-deoxy-d-xylulose-5-phosphate synthase et sa surproduction dans les plantes
EP1294913B1 (fr) Modification de la teneur en produits chimiques fins dans les organismes par modification genetique du chemin du shikimate
EP1212439B1 (fr) Plantes a teneur modifiee en acides amines et leur procede de production
DE19752647C1 (de) Reduktiion des Chlorophyllgehaltes in Ölpflanzensamen
DE10046462A1 (de) Verbesserte Verfahren zur Vitamin E Biosynthese
DE19918949A1 (de) Überexpression einer DNA-Sequenz codierend für eine 1-Desoxy-D-Xylulose-5-Phosphat Reduktoisomerase in Pflanzen
EP1194577A1 (fr) Identification et surexpression d'une sequence d'adn codant pour une 2-methyl-6-phytylhydroquinone-methyltransferase dans des plantes
WO1999029880A2 (fr) Procede de production de plantes transgeniques par biosynthese modifiee d'acide 5-aminolevulinique, et procede d'identification d'effecteurs de la synthese d'acide 5-aminolevulinique
DE19835219A1 (de) DNA-Sequenz codierend für eine 1-Deoxy-D-Xylulose-5-Phosphat Synthase und deren Überproduktion in Pflanzen
EP1198578A2 (fr) S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION
DE10111676A1 (de) Erhöhung des Vitamin-E-Gehalts in Organismen durch Erhöhung der Tyrosinaminotransferase-Aktivität
DE19903493A1 (de) Überexpression einer DNA-Sequenz codierend für eine Transketolase in Pflanzen
WO1999050400A1 (fr) Adenylique-desaminase
WO1999022011A1 (fr) Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses
WO2004058934A2 (fr) Procede pour produire des plantes transgeniques a teneur accrue en vitamine e par modification de la teneur en serine-acetyltransferase
EP1210437B1 (fr) Dihydro-orotase issue de vegetaux
DE19914792A1 (de) Metabolische Selektionsmarker für Pflanzen
DE102004036466B4 (de) Verfahren zur Herstellung von C6- und/oder C9-Aldehyden und -Alkoholen in Pflanzen durch 9/13-Divinylethersynthase
DE19845231A1 (de) DNA-Sequenzen codierend für eine 1-Deoxy-D-Xylulose-5-Pphosphat Synthase, eine Hydroxyphenylpyruvat Dioxygenase und eine Geranylgeranylpyrophosphat Oxidoreduktase und deren Überproduktion in Pflanzen
WO2001036606A2 (fr) Production de plantes resistant aux inhibiteurs peroxydants de la protoporphyrinogene-9-oxydase
DE19845216A1 (de) DNA-Sequenzen codierend für eine 1-Deoxy-D-Xylulose-5-Phosphat Synthase und eine Hydroxyphenylpyruvat Dioxygenase und deren Überproduktion in Pflanzen
DE19845224A1 (de) DNA-Sequenzen codierend für eine 1-Deoxy-D-Xylulose-5-Phosphat Synthase und eine Geranylgeranyl-Pyrosphat Oxidoreduktase und deren Überproduktion in Pflanzen
WO2001064861A2 (fr) Synthetase 1 de pyrophosphate de phosphoribosyle en tant que cible herbicide

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 2000958357

Country of ref document: EP

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWP Wipo information: published in national office

Ref document number: 2000958357

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2000958357

Country of ref document: EP