EP1255841A2 - Utilisation de sequences de palatinase et de trehalulase en tant que marqueurs nutritifs dans des cellules transformees - Google Patents

Utilisation de sequences de palatinase et de trehalulase en tant que marqueurs nutritifs dans des cellules transformees

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Publication number
EP1255841A2
EP1255841A2 EP01909747A EP01909747A EP1255841A2 EP 1255841 A2 EP1255841 A2 EP 1255841A2 EP 01909747 A EP01909747 A EP 01909747A EP 01909747 A EP01909747 A EP 01909747A EP 1255841 A2 EP1255841 A2 EP 1255841A2
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Prior art keywords
nucleic acid
protein
recombinant nucleic
dna
sequence
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EP01909747A
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German (de)
English (en)
Inventor
Frederik BÖRNKE
Uwe Sonnewald
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Institut fuer Pflanzengenetik und Kulturpflanzenforschung
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Institut fuer Pflanzengenetik und Kulturpflanzenforschung
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Priority claimed from DE10045182A external-priority patent/DE10045182A1/de
Application filed by Institut fuer Pflanzengenetik und Kulturpflanzenforschung filed Critical Institut fuer Pflanzengenetik und Kulturpflanzenforschung
Publication of EP1255841A2 publication Critical patent/EP1255841A2/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/27—Erwinia (G)
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • 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
    • C12N9/90—Isomerases (5.)

Definitions

  • the present invention relates to recombinant nucleic acid molecules which contain a DNA sequence which contains a protein with the enzymatic activity of a palatinose hydrolase. also called palatinase, or a trehalulose hydrolase, also called trehalulase, encodes, and the use of these nucleic acid molecules as selection markers in transformed cells.
  • the invention relates to recombinant nucleic acid molecules which contain a DNA sequence which encodes a protein with the enzymatic activity of a palatinase or a trehalulase, and in which this DNA sequence is under the control of regulatory sequences of a promoter active in plants.
  • the invention further relates to vectors and host cells which contain the recombinant nucleic acid molecules according to the invention. Methods for producing transformed plant cells and plants using the recombinant nucleic acid molecules and vectors are also provided.
  • the invention further relates to transgenic plants, their harvest products and propagation material and transgenic plant cells which contain the nucleic acid molecules or vectors according to the invention or are produced by the method according to the invention.
  • the invention also relates to the use of DNA sequences which encode a protein with the enzymatic activity of a palatinose hydrolase or a trehalulose hydrolase as a nutritional selection marker in plant cells for the selection of transformed plant cells and plants. Furthermore, the invention relates to a method for the selection of microorganisms on a medium containing palatinose or trehalulose.
  • Plant protection measures are: i) herbicide-tolerant plants, ii) insect-resistant, virus-resistant and fungus-resistant plants and iii) ozone-resistant plants.
  • quality increases are: i) increasing the shelf life of fruit, ii) increasing the starch production in potato tubers, iii) changing the carbohydrate and lipid composition and iv) producing non-plant polymers.
  • the basic prerequisite for the generation of transgenic plants is the availability of suitable transformation systems and the presence of selectable markers that enable the identification of successfully transformed plant cells.
  • the target gene that is to say, is produced in a conventional manner gene interested in agronomic view, a selectable marker gene integrated into the plant genome, which enables the identification of transgenic cells in a relatively simple and efficient manner.
  • Genes that mediate herbicide or antibiotic tolerance are currently used to select transformed plant cells. Suitable resistance genes are for example the bar gene from Streptomyces hygroscopicus which confers resistance to the herbicide phosphinotricin Total (BASTA ®) mediates (De Block et al (1987) EMBO J. 6:. 2513- 2518).
  • Further selection markers which are used in plant transformation impart resistance to G 418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea and gentamycin to the transformed plant cells.
  • Metabolic processes require the manipulation of several enzymatic steps. This means that the possibility of transforming transgenic plants several times is essential in the area of "metabolism modeling". The reasons mentioned have led to the search for further selectable markers being intensified. Despite intensive efforts, only a few new markers for the selection of transformed plant cells have been successfully used. For example, by expressing a mannose-6-phosphate isomerase, a positive selection for mannose-containing nutrient medium for transformed plant cells could be established. Another method takes advantage of the ability of an Aspergillus terreus deaminase to detoxify the insecticide Blasticidin S.
  • the present invention is therefore based on the object of providing a new marker for the selection of transformed plant cells and plants.
  • DNA sequences which encode a protein with the enzymatic activity of a palatinase or a trehalulase are suitable for the selection of transformed plant cells and plants.
  • the selection according to the invention on or in medium containing palatinose or medium containing trehalulose does not lead to the death of untransformed cells.
  • the transformed cells according to the invention can metabolize the palatinose or trehalulose and thus have a selection advantage.
  • mannose system mentioned above.
  • mannose marker cannot be used in all crop plants.
  • the invention thus relates to a recombinant nucleic acid molecule, comprising a) regulatory sequences of a promoter active in plant cells; b) operatively linked to a DNA sequence that encodes a protein with the enzymatic activity of a palatinase or a trehalulase: and c) operatively linked to regulatory sequences that are used as transcription, termination and / or polyadenylation signals in
  • Plant cells can serve.
  • a protein with the enzymatic activity of a palatinase or trehalulase is understood to be a protein which is able to cleave palatinose or trehalulose, in particular that
  • disaccharide palatinose or trehalulose to catalyze the monosaccharides fructose and glucose.
  • the two monosaccharide units are present in an ⁇ 1-6 glycosidic bond, while in trehalulose fructose and glucose are linked via a cd> glycosidic bond.
  • PCT / EP 95/00165 discloses the sequence of a palatinase gene from the Protaminobacter rubrum bacterium and the sequence of a palatinase gene from the Pseudomonas mesoacidophila MX-45 bacterium.
  • Nucleic acid molecules in which a DNA sequence encoding a palatinase is under the control of a plant promoter, and the expression of a protein with palatinase activity in plants are just as new as the DNA sequence from Erwinia rhapontici, which has now been disclosed for the first time in the context of this invention
  • Protein encoded with the enzymatic activity of a palatinase This sequence is in the attached sequence listing under SEQ ID No. 1, the deduced amino acid sequence is given under SEQ ID No. 2 and SEQ ID No. 3 specified.
  • a DNA sequence which encodes a protein with the enzymatic activity of a trehalulase.
  • SEQ ID No. 25 the deduced amino acid sequence is given under SEQ ID No. 26 and 27 respectively.
  • the invention thus also relates to those in SEQ ID No. 1 or SEQ ID No. 25 specified nucleotide sequences which encode a protein with the enzymatic activity of a palatinase or trehalulase, and the use of
  • Nucleic acid molecules encode the proteins with the enzymatic activity of a palatinase or trehalulase, for the selection of transformed plant cells and plants.
  • transformed plant cells or plants which express a palatinase or trehalulase can grow and regenerate on or in medium containing palatinose or trehalulose due to this new enzymatic property, while wild-type plant cells or plants are not able to utilize palatinose or trehalulose as a carbohydrate source and therefore neither grow nor regenerate on or in medium containing palatinose or trehalulose.
  • This observation can be used ideally for the selection of transformed plant cells and plants.
  • Palatinase and trehalulase can be used as new nutritional markers in all plants that naturally cannot convert and utilize the disaccharides palatinose or trehalulose.
  • the present invention comprises both the possibility of transforming plants alternatively with a palatinase gene or a trehalulose gene and the possibility of transforming plants with a palatinase gene and with a trehalulase gene.
  • palatinase sequences or trehalulase sequences as a nutritional selection marker, which has now been observed for plant cells for the first time, can also be successfully used for other transformed cells.
  • the principle according to the invention of utilizing the non-usable sugars palatinose or trehalulose can be applied to any cell or organism that is naturally unable to utilize palatinose or trehalulose.
  • the invention thus also relates to the use of DNA sequences which encode a protein with the enzymatic activity of a palatinase or trehalulase, as a marker for the selection of transformed cells which are not plant cells but other eukaryotic or prokaryotic cells or Organisms. These are preferably microorganisms such as bacteria. Viruses, fungi, yeasts and algae.
  • a secreted palatinase or trehalulase can be expressed in E. coli, for example, and successfully transformed bacterial cells can then be selected on medium containing palatinose or trehalulose.
  • the specialist is familiar with the cloning, expression and transformation techniques required for this.
  • the palatinase can also be expressed inside the cell of the cell to be selected, e.g. in the cytosol of an E. coli cell. This has the advantage that neither the palatinase nor the hexoses resulting from the palatinase activity can diffuse into the medium.
  • the additional expression of the palatinose transporter isolated and characterized for the first time in the context of this invention lends itself. The above statements apply analogously to the expression of a trehalulase.
  • palatinose operon was cloned from Erwinia rhapontici in the context of this invention, comprising the following genes:
  • the transfer of the entire operon or at least the genes palE, palF, palG and palK involved in the ABC transport system leads to the expression of the palatinose transporter in the transformed cells, preferably microorganisms.
  • the cells expressing the transporter can take up the palatinose or trehalulose from the medium, so that the hydrolysis by the selection marker palatinase or trehalulase can take place in the cytosol of the cell.
  • DNA sequence encoding a protein with the enzymatic activity of a palatinase or trehalulase can be isolated from natural sources or synthesized by conventional methods. Using common molecular biological techniques (see for example Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) it is possible to prepare or produce desired constructs for the transformation of plant cells. The cloning, mutagenizing, sequence analysis, and commonly used for genetic engineering manipulation in prokaryonti cells
  • deletion mutants in which the synthesis of correspondingly shortened proteins can be achieved by progressive deletion from the 5 'or from the 3' end of the coding DNA sequence.
  • enzymes that are localized in certain compartments of the plant cell by adding corresponding signal sequences.
  • Such sequences are described in the literature and are well known to those of ordinary skill in the art (see, for example, Braun et al. (1992) EMBO J. 11: 3219-3227; Wolter F. et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 846-850; Sonnewald U. et al. (1991) Plant J. 1: 95-106).
  • mutants can be produced which have an altered substrate or product specificity. Furthermore, mutants can be produced which have a changed activity, temperature and / or pH profile.
  • nucleic acid molecules according to the invention or parts thereof can be used in the genetic engineering manipulation in prokaryotic cells.
  • the recombinant nucleic acid molecules according to the invention or parts thereof can be used in the genetic engineering manipulation in prokaryotic cells.
  • Plasmids are introduced which allow mutagenesis or a sequence change by recombining DNA sequences. With the help of standard procedures (see e.g. Sambrook et al. (1989), vide supra), base exchanges can be made or natural or synthetic sequences can be added. To connect the DNA fragments to one another, adapters or linkers can be added to the fragments where necessary. Appropriate restriction sites can also be provided by means of enzymatic and other manipulations, or superfluous DNA or restriction sites can be removed. Where insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. Sequence analysis, restriction analysis and other biochemical-molecular biological methods are generally used as analysis methods.
  • the person skilled in the art can find further DNA sequences which encode proteins with palatinase or trehalulase activity from other organisms by means of conventional molecular biological techniques and use them in the context of the present invention.
  • the person skilled in the art can derive suitable hybridization probes from the known palatinase sequences and use them for the screening of cDNA and / or genomic banks of the desired organism from which a new palatinase gene is to be isolated.
  • the person skilled in the art can fall back on common hybridization, cloning and sequencing methods which are well known and established in every biotechnological or genetic engineering laboratory (see, for example, Sambrook et al (1989). Vide supra).
  • the DNA sequence which encodes a protein with the enzymatic activity of a palatinase is selected from the group consisting of a) DNA sequences which comprise a nucleotide sequence which has the sequence shown in SEQ ID NO. 3 encode specified amino acid sequence or fragments thereof, b) DNA sequences which contain the sequence shown in SEQ ID No. 1 specified nucleotide sequence or parts thereof, c) DNA sequences comprising a nucleotide sequence.
  • DNA sequences which comprise a nucleotide sequence which has been degenerated to a nucleotide sequence of c), or comprise parts of this nucleotide sequence e) DNA - Sequences that are a derivative, analogue or fragment of a nucleotide sequence of a). b). c) or d).
  • the DNA sequence encoding a protein with the enzymatic activity of a palatinase is selected from those in PCT / EP 95/00165 in SEQ ID No. 7 and SEQ ID No. 15 indicated DNA sequences as well as DNA sequences which comprise a nucleic acid sequence which hybridize with a complementary strand of these DNA sequences, as well as DNA sequences which comprise a nucleic acid sequence which is degenerate to one of the aforementioned nucleic acid sequences and DNA sequences, which represent a derivative, analog or fragment of one of the preceding nucleic acid sequences and encode a protein with palatinase activity.
  • the DNA sequence encoding a protein with the enzymatic activity of a trehalulase is selected from the group consisting of
  • DNA sequences which comprise a nucleotide sequence which the in SEQ ID NO. 27 encode the amino acid sequence indicated or fragments thereof b) DNA sequences which the SEQ ID No. 25 specified nucleotide sequence or parts thereof, c) DNA sequences which comprise a nucleotide sequence which hybridizes with a complementary strand of the nucleotide sequence of a) or b), or parts thereof
  • Nucleotide sequence comprise, d) DNA sequences which comprise a nucleotide sequence which is degenerate to a nucleotide sequence of c) or parts of this nucleotide sequence, e) DNA sequences which are a derivative, analog or fragment of a nucleotide sequence of a). b), c) or d).
  • a DNA sequence is thus provided which encodes a protein with the enzymatic activity of a palatinase and / or a trehalulase, wherein in the protein with the enzymatic activity of a palatinase and / or the protein with the enzymatic Activity of a trehalulase is inactivated by at least one amino acid exchange for the wild protein a glycosylation site.
  • hybridization means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al. (1989. vide supra).
  • the stringent conditions are particularly preferred as follows: 1 x SSC, 0.1% SDS at a hybridization temperature of 55 ° C. for a period of 1 h.
  • DNA sequences that hybridize with the DNA sequences that encode a protein with the enzymatic activity of a palatinase or trehalulase can, for example, from genomic or cDNA libraries of any organism, the palatinase DNA sequences or trehalulase DNA Sequences naturally has to be isolated. Such DNA sequences can be identified and isolated, for example, using DNA sequences which are exactly or essentially those in SEQ ID No. 1 or SEQ ID No.
  • the fragments used as the hybridization probe can also be synthetic fragments which have been produced with the aid of conventional synthesis techniques and whose sequence essentially corresponds to one of the abovementioned palatinase DNA sequences or trehalulase DNA sequences or a part thereof.
  • the DNA sequences which encode a protein with the enzymatic activity of a palatinase or trehalulase also comprise DNA sequences whose nucleotide sequences are degenerate to that of one of the DNA sequences described above.
  • the degeneration of the genetic code offers the person skilled in the art the possibility, among other things, of adapting the nucleotide sequence of the DNA sequence to the codon preference (codon usage) of the target plant, ie the adapt the selectable plant or plant cell based on the expression of the palatinase DNA sequence on or in the palatinose-containing medium and thereby optimize the expression.
  • the DNA sequences described above also include fragments, derivatives and allelic variants of the DNA sequences described above, which encode a protein with the enzymatic activity of a palatinase or trehalulase.
  • “Fragments” are understood to mean parts of the DNA sequence that are long enough to encode one of the proteins described.
  • the term “derivative” in this context means that the sequences differ from the DNA sequences described above in one or more positions, but have a high degree of homology to these sequences.
  • Homology means a sequence identity of at least 40 percent, in particular an identity of at least 60 percent, preferably over 80 percent and particularly preferably over 90 percent.
  • the proteins encoded by these DNA sequences have a sequence identity to that in SEQ ID No. 2 and 3 or SEQ ID No.
  • 26 and 27 indicated amino acid sequence of at least 80 percent, preferably 85 percent and particularly preferably over 90 percent, 95 percent and 98 percent.
  • the deviations from the DNA sequences described above can be caused, for example, by deletion. Substitution, insertion or recombination have arisen.
  • DNA sequences which are homologous to the sequences described above and which are derivatives of these sequences are generally variations of these sequences which are modifications which have the same biological function. These can be both naturally occurring variations, for example sequences from other organisms, or mutations, these mutations occurring naturally may have occurred or were introduced through targeted mutagenesis. Furthermore, the variations can be synthetically produced sequences.
  • allelic variants can be both naturally occurring variants and also synthetically produced variants or those produced by recombinant DNA techniques. The above, in connection with palatinase or
  • Trehalulase DNA sequences also apply to the other Erwinia rhapontici DNA sequences made available for the first time in the context of this invention.
  • the described DNA sequences coding for a palatinase or trehalulase originate from Erwinia rhapontici.
  • the promoter can be selected so that the expression is constitutive or only in a certain tissue or organ, at a certain time in plant development and / or at a time determined by external influences, biotic or abiotic stimuli (induced gene expression).
  • the promoter can be homologous or heterologous with respect to the plant to be transformed.
  • Suitable promoters are, for example, the 35S RNA promoter of the Cauliflower Mosaic Virus and the ubiquitin promoter from maize for constitutive expression, a promoter which is only expressed in photosynthetically active tissues guaranteed.
  • the ST-LS1 promoter Stockhaus et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7943-7947; Stockhaus et al. (1989) EMBO J. 8: 2445-2451
  • a promoter which is active during plant transformation, plant regeneration or certain stages of these processes, such as cell division-specific promoters such as the histone H3 promoter (Kapros et al. (1993) InVitro Cell Cev.
  • the promoter is the 35S RNA promoter from CaMV.
  • 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 invention further relates to vectors which contain nucleic acid molecules according to the invention and whose use enables the selection of transformed plant cells and plants.
  • vectors which contain nucleic acid molecules according to the invention and whose use enables the selection of transformed plant cells and plants.
  • These are in particular plasmids, cosmids. Viruses, bacteriophages and other vectors commonly used in genetic engineering.
  • the desired sequence can be introduced into the vector at a suitable restriction site.
  • the plasmid obtained is then used for the transformation of E. co / z ' cells.
  • Transformed E. coli cells are grown in an appropriate medium and then harvested and lysed, and the plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analytical method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences.
  • the recombinant nucleic acid molecule according to the invention which contains a DNA sequence encoding a palatinase or trehalulase, or the vector according to the invention, containing a nucleic acid molecule according to the invention with a palatinase or trehalulase DNA sequence, contains at least one further recombinant nucleic acid molecule, that carries any genetic information that is to be transferred together with the palatinase DNA sequence to plant cells or plants.
  • any information contained in the further recombinant nucleic acid molecule can be obtained.
  • the additional genetic information which is to be transferred to plant cells does not necessarily have to be present on the same vector together with the selection markers palatinase and trehalulase. Rather, the nucleic acid molecule that bears the selection marker on the one hand and the nucleic acid molecule that bears the additional information on the other hand can also be introduced into a plant cell separately, so-called co-transformation. This procedure is particularly useful in cases where a physical coupling of the marker gene and the information to be transmitted is not desired. In this case, after the selection of the primary transformants, the marker gene and the desired information can segregate independently of one another in subsequent crossings.
  • the further recombinant nucleic acid molecule contains a DNA sequence which encodes a peptide, a protein, an antisense RNA, a sense RNA, a viral RNA or a ribozyme.
  • Herbers and Sonnewald (1996, TIBTECH 14: 198-205) summarize some examples of heterologous expression or overexpression and of antisense inhibition with the aim of manipulating metabolic pathways in transgenic plants. Other goals are in particular the transfer of resistance, the use of plants as
  • Production facility for proteins Carbohydrates, fats, renewable raw materials and other substances that can be synthesized by plants.
  • the vectors and nucleic acid molecules according to the invention can have further regulatory sequences and / or functional units which e.g. as
  • Enhancers act or stabilize the vector in the host cell.
  • the coding sequences can also be signal sequences be supplemented, which ensure the transport of the gene product, in this case the protein with palatinase or trehalulase activity, to a specific compartment.
  • signal sequences ensure that the palatinase or trehalulase is transformed into the cell wall of the
  • Plant cells is transported. i.e. the transformed plants express a chimeric palatinase or trehalulase. which comprises a signal peptide for transport into the cell wall.
  • the respective enzyme cleaves the palatinose or trehalulose offered in the medium into glucose and fructose, which are taken up and metabolized in the cell.
  • Suitable signal sequences which ensure inclusion in the endoplasmic reticulum can be found in the relevant literature by the person skilled in the art.
  • sequence coding for the signal peptide of the proteinase inhibitor II gene from potato is particularly suitable (Keil et al. (1996) Nucl. Acids Res. 14: 5641-5650; Genbank Accession No. X041 18).
  • the invention also relates to host cells which contain the recombinant nucleic acid molecules or vectors according to the invention, it being possible for the molecules or vectors to be transient or stable. Every cell and everyone can host cells
  • prokaryotic and eukaryotic cells or organisms come into question, in particular microorganisms such as bacteria.
  • Viruses Fungi, yeasts and algae, but also plant cells, which contain the nucleic acid molecules according to the invention or vectors or parts or derivatives thereof.
  • plant cells are preferably a palatinase or trehalulase due to the absorption of the invention coding DNA sequences able to synthesize proteins with the enzymatic activity of a palatinase or trehalulase.
  • kits which i) contain a recombinant nucleic acid molecule or a vector according to the invention, the nucleic acid molecule or the vector can also be present in a host cell, and ii) optionally contain palatinose or a carbohydrate equivalent to palatinose.
  • the carbohydrate equivalent to palatinose can generally be a disaccharide in which a fructose and a glucose molecule are linked via an ⁇ -glycosidic bond, such as, for example, turanose (3-O- ⁇ -D-glucopyranosyl-D-fructose ) or trehalulose (1-O- ⁇ -D-glucopyranosyl-D-fructose).
  • turanose 3-O- ⁇ -D-glucopyranosyl-D-fructose
  • trehalulose 1-O- ⁇ -D-glucopyranosyl-D-fructose
  • the present invention thus also relates to a method for selecting transformed plant cells, comprising the steps: i) introduction of a recombinant nucleic acid molecule, comprising a) regulatory sequences of a promoter active in plant cells; b) operatively linked to it a DNA sequence which encodes a protein with the enzymatic activity of a palatinase or trehalulase; and c) operationally linked regulatory sequences which can serve as transcription, termination and / or polyadenylation signals in plant cells, or a vector containing such a recombinant nucleic acid molecule in plant cells; and ii) selection of the transformed plant cells on or in palatinose or trehalulose or a medium containing carbohydrate equivalent to palatinose.
  • a prerequisite for the introduction of the recombinant nucleic acid molecules and vectors according to the invention in plant cells is the availability of suitable transformation systems.
  • transformation methods have been developed and established here over the past two decades. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, diffusion of protoplasts, the direct gene transfer of isolated DNA to protoplasts, the injection and electroporation of DNA into plant cells, the introduction of DNA by means of the biolistic Methods and other possibilities, whereby the person skilled in the art can easily determine the most suitable method. All transformation processes have been well established for many years and are undoubtedly part of the standard repertoire of the specialist in plant molecular biology, plant biotechnology and cell and tissue culture.
  • plasmids When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies to direct gene transfer. Simple plasmids, such as pUC derivatives, can be used. However, whole plants should be made from cells transformed in this way the presence of a selectable marker gene is recommended.
  • the plant cell uses the Ti or Ri plasmid, 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.
  • Agrobacteria are used for the transformation, 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 into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria.
  • 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.
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension cultivated
  • Plant cells can then be regenerated again in a suitable medium containing palatinose or trehalulose or an equivalent carbohydrate for the selection of transformed plant cells.
  • the selection medium should generally contain at least 0.1% palatinose or trehalulose, if possible between 0.1 and 3.0% palatinose or trehalulose.
  • the palatinose or trehalulose concentration is preferably between 0.8 and 2.4% and particularly preferably between 1.0 and 2.0%.
  • a palatinose or trehalulose content in the medium of 1.6% is most preferred.
  • Palatinose or trehalulose as the selection media are the only carbon sources in the medium.
  • the regeneration of the transgenic plants from transgenic plant cells is carried out according to customary regeneration methods using conventional nutrient medium and phytohormones, with the difference that palatinose or trehalulose is present as a selection agent.
  • sequence-specific recombinases can be used, for example in the form of the retransformation of a starting line expressing recombinase and outcrossing of the recombinase after removal of the Selection markers (see e.g. 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.
  • the invention further relates to plant cells which contain a nucleic acid molecule according to the invention or a vector according to the invention with palatinase or trehalulase DNA sequences.
  • the plant cell according to the invention contains at least one further foreign gene.
  • the invention also relates to the transgenic plants obtainable by regeneration of the plant cells according to the invention.
  • the transgenic plant or the transgenic plant cells can be any monocot or dicot plant or plant cell, preferably it is useful plants or cells of useful plants.
  • the invention also relates to harvest products and propagation material of transgenic plants, the cells or tissues of which contain a nucleic acid molecule according to the invention.
  • the harvested products and the propagation material are in particular fruits, seeds, tubers, rhizomes, seedlings, cuttings, etc. If the expression of the palatinase or trehalulase in the transgenic plant cells and plants is under the control of a constitutive promoter, the plants, parts of plants, harvested products and propagation material can also utilize the ability to utilize palatinose or trehalulose and therefore on or in palatinose-containing Medium or trehalulose-containing medium to be identified.
  • palatinase or trehalulase DNA sequence is under the control of, for example, an inducible or cell or tissue-specific promoter
  • the plants, parts of plants, harvested products and propagation material may have to be identified by means of molecular biological or biochemical methods.
  • a broad spectrum of molecular biological and / or biochemical methods is available to the person skilled in the art for the analysis of the transformed plant cells, transgenic plants, plant parts, crop products and propagation material , eg PCR, Northern blot analysis for the detection of palatinase-specific RNA or for determining the level of accumulation of palatinase-specific RNA, southern blot analysis for the identification of palatinase-coding DNA sequences or Western blot analysis for the detection of the nucleic acid molecules according to the invention encoded palatinase.
  • PCR Northern blot analysis for the detection of palatinase-specific RNA or for determining the level of accumulation of palatinase-specific RNA
  • southern blot analysis for the identification of palatinase-coding DNA sequences
  • Western blot analysis for the detection of the nucleic acid molecules according to the invention encoded palatinase.
  • the detection of the enzymatic activity of palatinase can also be determined by a person skilled in the art using protocols available in the literature.
  • the seeds obtained by self-cultivation or crossings can be laid out on medium containing palatinose and conclusions can be drawn on the genotype of the respective plant based on the germination capacity and the growth of the daughter generation (s) and the segregation pattern.
  • the above statements apply analogously to the analysis of plant cells and plants transformed with trehalulase DNA sequences.
  • the invention also relates to the use of DNA sequences which encode a protein with the enzymatic activity of a palatinase or trehalulase as a selection marker in plant transformation or cell and tissue culture.
  • a recombinant nucleic acid molecule or vector according to the invention is used as a selection marker in the production of transgenic plant cells and plants.
  • the invention is based on the observation that DNA sequences which encode a protein with the enzymatic activity of a palatinase or trehalulase can be used as selection markers in plant transformation, which is shown in the following examples, which serve only to illustrate the invention and in are in no way to be understood as a limitation.
  • Cloning methods such as: restriction cleavage, DNA isolation, 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, sequence analysis of recombinant DNA were carried out according to Sambrook et al (1989. vide supra). The transformation of Agrobacterium tumefaciens was carried out according to the Höfgen and Willmitzer method (1988. Nucl. Acids Res. 16: 9877). Agrobacteria were grown in YEB medium (Vervliet et al. (1975) Gen. Virol. 26:33).
  • chromosomal DNA was isolated from the cells of a 50 ml overnight culture according to the standard protocol. Then approximately 300 ⁇ g of the DNA was partially digested with the restriction enzyme Sau3A and separated on a preparative agarose gel. Fragments between 5 and 12 kb were eluted from the gel using the Qiaquick Gel Extraction Kit (Qiagen, Hilden). The DNA fragments obtained in this way were ligated into BamHI-digested Lambda ZAP-Express arms (Stratagene, La Jolla, USA) and then packaged in vitro (Gigapack III Gold Packaging Extract, Stratagene, according to the manufacturer's instructions). E. co // bacteria of the strain XL-MRF '(Stratagene) were infected with recombinant lambda phages, the titer of the bank determined and finally the bank amplified.
  • E. coli Bacterial strains and plasmids E. coli (XL-1 Blue, XL-MRF 'and XLOLR) bacteria were obtained from Stratagene. Erwinia rhapontici (DSM 4484) was obtained from the German Collection for Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). The Agrobacterium used for plant transformation Strain (C58C1 with plasmid pGV 3850kan) was developed by Debleare et al. (1985, Nucl. Acids Res. 13: 4777).
  • the vectors pCR-Blunt (Invitrogen, Netherlands), pMAL-c2 (New England Biolabs), pUC 19 (Yanish-Perron (1985) Gene 33: 103-1 19) and Binl9 (Bevan (1984) Nucl. Acids Res. 12: 871 1-8720) was used.
  • sucrose isomerase A subfragment of sucrose isomerase was cloned by means of polymerase chain reaction (PCR). Genomic DNA from E. rhapontici, which according to Standard protocol was isolated. The amplification was carried out using the specific primers
  • Primer FB 84 5'-GTCGACGTCTTGCCAAAAACCTT-3 ', which were derived from a sucrose isomerase sequence of the prior art.
  • Primer FB 83 comprises bases 109-127 and primer FB 84 bases 1289-1306 of the coding region of the sucrose isomerase gene from E. rhapontici.
  • the PCR reaction mixture (100 ⁇ l) contained chromosomal bacteria DNA (1 ⁇ g), primers FB 83 and FB 84 (250 ng each), Pfu DNA polymerase reaction buffer (10 ⁇ l, Stratagene), 200 ⁇ M dNTPs (dATP, dCTP, dGTP, dTTP) and 2.5 units of Pfu DNA polymerase (Stratagene).
  • the mixture was heated to 95 ° C. for 5 min.
  • the polymerization steps (30 cycles) were carried out in an automatic T3 thermal cycler (Biometra) according to the following program: denaturation 95 ° C (1 minute), attachment of the primers at 55 ° C (40 seconds), polymerase reaction at 72 ° C ( 2 minutes).
  • the fragment obtained was cloned into the vector pCR blunt (Invitrogen). The identity of the amplified DNA was verified by sequence analysis.
  • Example 2 To isolate the palatinose operon, a genomic library with a subfragment of sucrose isomerase (see Example 1) was screened. Several positive clones were isolated. By completely sequencing and assembling these clones, several open reading frames could be identified which code for enzymes of the palatinose metabolism (see above overview of the genes of the palatinose operon and the associated gene products).
  • the sketch below shows a schematic overview of the cloned palatinose gene cluster from Erwinia rhapontici. The position of the open reading frame and the direction of transcription are shown by arrows.
  • the complete open reading frame of the palatinase was cloned by means of the polymerase chain reaction (PCR). Genomic DNA from E. rhapontici, which was isolated according to the standard protocol, was used as template material. The amplification was carried out using the specific primers
  • Primer FB 180 comprises bases 2-21, primer FB 176 bases 1638-1656 of the coding region of the palatinase gene.
  • the primers additionally carry the following restriction cloning sites: Primer FB 180 Bglll; Primer FB 176 Sall.
  • the PCR reaction mixture (100 ⁇ l) contained chromosomal bacterial DNA (1 ⁇ g), primers FB 180 and FB 176 (250 ng each), Pfu DNA polymerase reaction buffer (10 ⁇ l, Stratagene), 200 ⁇ M dNTPs (dATP, dCTP, dGTP.dTTP) and 2.5 units of Pfu DNA polymerase (Stratagene).
  • the DNA sequence which codes for a palatinase, was fused with a signal peptide of a plant gene (proteinase inhibitor II gene from potato (Solanum tuberosum, Keil et al (1986, vide supra)) required for inclusion in the endoplasmic reticulum) and under brought the control of the 35S RNA promoter, whereby the plasmid p35S-cwp ⁇ / ⁇ ) was formed.
  • a plant gene proteinase inhibitor II gene from potato (Solanum tuberosum, Keil et al (1986, vide supra) required for inclusion in the endoplasmic reticulum
  • the palatinase fragment was cut out of the construct pCR-palQ via the restriction sites BglII and Sall and ligated into a pMA vector opened in BamHI / Sall.
  • the vector pMA represents a modified form of the vector pBinAR (Höfgen and Willmitzer (1990) Plant Sci.66: 221-230), which contains the 35S promoter of the Cauliflower mosaic virus, which mediates constitutive expression in transgenic plants Signal peptide of the proteinase inhibitor II from potato (Keil et al. 1986, vide supra)), which mediates the targeting of the fusion protein into the cell wall, and contains a plant termination signal.
  • the plant termination signal includes the 3 'end of the polyadenylation site of the octopine synthase gene. Interfaces for the restriction enzymes BamHI are located between the partial sequence of the proteinase inhibitors and the termination signal.
  • Xbal, Sall, PstI and SphI in this order, which allow the insertion of corresponding DNA fragments, so that a fusion protein is formed between the proteinase inhibitor and the inserted protein, which is then in the cell wall of transgenic plants or plant cells that express this protein (see Figure 2).
  • Fragment A contains the Cauliflower Mosaic Virus (CaMV) 35S RNA promoter. It contains a fragment which comprises nucleotides 6909 to 7437 of the CaMV (Franck (1980) Cell 21, 285.
  • CaMV Cauliflower Mosaic Virus
  • Fragment B contains nucleotides 923-1059 of a proteinase inhibitor II gene from the potato (Solanum tuberosum, Keil et al. 1986, vide supra), which targets the palatinase gene via a linker with the sequence ACC GAA TTG GG Erwinia rhapontici, which comprises nucleotides 2-1656, is fused.
  • a signal peptide of a vegetable protein necessary for the uptake of proteins into the endoplasmic reticulum is fused to the palatinase sequence at the N-terminal.
  • Fragment C contains the polyadenylation signal of gene 3 of the T-DNA of the ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3, 835), nucleotides 11749-11939.
  • the functional characterization of the palatinase gene was carried out by expression of the recombinant protein in E. coli.
  • the plasmid pQE-palQ was transformed into E. coli (XL-I blue, Stratagene).
  • the recombinant protein was expressed in one according to the manufacturer's instructions (Qiagen, Hilden, Germany) Culture scale of 50 ml. After harvesting the cells by centrifugation, the pellet was resuspended in 1 ml of 30 mM HEPES (pH 7.5) and the soluble protein fraction was released by ultrasound treatment. 20 ⁇ l of the crude extract were mixed with 80 ⁇ l 100 mM palatinose and incubated at 30 ° C. for 40 minutes. To detect the palatinase activity, the glucose released was determined in an aliquot of the mixture by a coupled optical-enzymatic test. The palatinase activity of the recombinant enzyme was clearly demonstrated.
  • the enzyme shows its highest activity at a reaction temperature of 30 ° C and a pH of 7.0.
  • a K m value for palatinose of 10 mM and a maximum reaction rate for a substrate concentration of 90 mM palatinose could be determined.
  • Figure 3 shows the cultivation of tobacco leaf discs on MS medium with palatinose as the sole carbon source (Fig. 3A: with 0.8% palatinose.
  • Fig. 3B with 1.6% palatinose
  • leaf disks do not form calli on a medium with palatinose as the only carbon source.
  • the addition of palatinose has no toxic effect on the plant cells.
  • tobacco was transformed using Agrobacterium-mediated gene transfer with the construct p35S-cwPal (see Example 4) (leaf disk transformation) and the leaf disks following the infection and incubation with Agrobacteria on MS regeneration medium with 1.6% palatinose cultivated as the only carbon source. After about 3 weeks, transformed shoots were visible, which were rooted with palatinose on hormone-free MS medium.
  • the complete open reading frame of the trehalulase was cloned by means of the polymerase chain reaction (PCR). Genomic DNA from E. rhapontici, which was isolated according to the standard protocol, was used as template material. The amplification was carried out using the specific primers
  • Primers FBI 84 and FBI 85 comprise bases 4-23 and 1638-1659, respectively, of the coding region of the trehalulase gene.
  • the primers additionally carry the following restriction cloning sites: primers FB 96 or FBI 84, BamHI; primer
  • the PCR reaction mixture (100 ⁇ l) contained chromosomal bacteria
  • DNA (1 ⁇ g), primers FB184 and FB185 (each 250 ng), Pfu DNA polymerase reaction buffer (10 ⁇ l, Stratagene), 200 ⁇ M dNTPs (dATP, dCTP, dGTP, dTTP) and 2.5 units Pfu DNA polymerase (Stratagene).
  • Pfu DNA polymerase reaction buffer 10 ⁇ l, Stratagene
  • 200 ⁇ M dNTPs dATP, dCTP, dGTP, dTTP
  • 2.5 units Pfu DNA polymerase (Stratagene).
  • the polymerization steps (30 cycles) were carried out in an automatic T3 thermal cycler (Biometra) according to the following program: denaturation 95 ° C (1 minute), attachment of the primers at 55 ° C (40 seconds), polymerase reaction at 72 ° C ( 2 minutes).
  • Fragment A contains the sequence of an E. rhapontici trehalulase that extends from nucleotide 4 - 1659 of the trehalulase gene.
  • the DNA sequence which codes for a trehalulase, was fused with a signal peptide of a plant gene (proteinase inhibitor II gene from potato (Solanum tuberosum, Keil et al. 1986, vide supra)) necessary for inclusion in the endoplasmic reticulum and under brought the control of the 35S RNA promoter, whereby the plasmid p35 S-cwpalZ was formed.
  • a plant gene proteinase inhibitor II gene from potato (Solanum tuberosum, Keil et al. 1986, vide supra)
  • the trehalulase fragment was cut out of the construct pCR-palZ via the restriction sites BamHI and Sall and ligated into a pMA vector opened in BamHI / Sall.
  • the pMA represents a modified form of the vector pBinAR (Höfgen and Willmitzer 1990, vide supra), which is the 35S promoter of the Cauliflower mosaic virus, which mediates constitutive expression in transgenic plants, a signal peptide of the proteinase inhibitor II from potato (Keil et al. 1986, vide supra), which mediates the targeting of the fusion protein into the cell wall, and contains a plant termination signal.
  • the plant termination signal contains the 3 'end of the polyadenylation site of the octopine synthase gene. Between the partial sequence of the proteinase inhibitor and the termination signal there are interfaces for restriction enzymes BamHI, Xbal, Sall, PstI and SphI (in this order), which allow the insertion of corresponding DNA fragments, so that a fusion protein between the proteinase inhibitor and the pasted Protein is formed, which is then transported into the cell wall of transgenic plants or plant cells that express this protein.
  • restriction enzymes BamHI, Xbal, Sall, PstI and SphI in this order
  • Fragment A contains the 35S promoter of the Cauliflower Mosaic Virus (CaMV). It contains a fragment which comprises the nucleotides 6909 to 7437 of the CaMV (Franck (1980) Cell 21: 285).
  • CaMV Cauliflower Mosaic Virus
  • Fragment B contains nucleotides 923-1059 of a proteinase inhibitor II gene from the potato (Solanum tuberosum) (Keil et al. 1986, vide supra), which targets the trehalulase gene via a linker with the sequence ACC GAA TTG GG
  • Erwinia rhapontici which comprises nucleotides 4 - 1659, is fused.
  • a signal peptide of a vegetable protein necessary for the uptake of proteins into the endoplasmic reticulum is fused to the trehalulase sequence at the N-terminal.
  • Fragment C contains the polyadenylation signal of gene 3 of the T-DNA of the ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3, 835), nucleotides 1 1749-1 1939.
  • pQE-palO corresponds to what the palatinase sequence relates to pCK-palQ, but is suitable for the expression of the palatinase sequence in E. coli.
  • the reaction mixture (50 ⁇ l) for PCR-based mutagenesis was composed as follows: 50 ng pQE-palO DNA, each 250 ng 5′- or 3′-primer, Pfu-DNA polymerase reaction buffer (5 ⁇ l.
  • the mutation event was verified in each case by sequencing the corresponding region of the palatinase sequence. Functional expression of the mutagenized enzyme in E. coli has shown in all cases that the respective amino acid substitution does not have a negative effect on the enzymatic activity. The mutations were then combined with one another using the strategy described above, so that ultimately a palatinase was produced which no longer has any putative glycosylation sites. This enzyme, too, had no adverse catalytic properties after expression in E. coli.
  • the mutated palatinase sequence was then cloned into a plant transformation vector and expressed in plants.

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Abstract

La présente invention concerne des molécules d'acides nucléiques recombinantes contenant une séquence d'ADN codant une protéine ayant l'activité enzymatique d'une hydrolase de palatinose, également connue sous le nom de palatinase, ou d'une hydrolase de tréhalulose, également connue sous le nom de tréhalulase. L'invention concerne également l'utilisation de ces molécules d'acides nucléiques en tant que marqueurs de sélection dans des cellules transformées. L'invention concerne en particulier des molécules d'acides nucléiques recombinantes contenant une séquence d'ADN codant une protéine ayant l'activité enzymatique d'une palatinase ou d'une tréhalulase, cette séquence d'ADN se trouvant sous le contrôle de séquences régulatrices d'un promoteur actif dans des plantes. L'invention concerne également des vecteurs et des cellules hôtes contenant les molécules d'acides nucléiques recombinantes selon l'invention, et des procédés de fabrication de cellules végétales transformées et de plantes au moyen des molécules d'acides nucléiques et vecteurs recombinants. L'invention concerne également des plantes transgéniques, leurs produits de récolte et substance de reproduction, ainsi que des cellules végétales transgéniques contenant les molécules d'acides nucléiques ou vecteurs selon l'invention ou fabriqués d'après le procédé selon l'invention. L'invention concerne également en particulier l'utilisation de séquences d'ADN codant une protéine ayant l'activité enzymatique d'une hydrolase de palatinose ou d'une hydrolase de tréhalulose, en tant que marqueurs de sélection nutritifs dans des cellules végétales pour la sélection de cellules végétales et de plantes transformées. L'invention concerne également un procédé de sélection de micro-organismes par rapport à un milieu contenant du palatinose ou du tréhalulose.
EP01909747A 2000-02-14 2001-02-14 Utilisation de sequences de palatinase et de trehalulase en tant que marqueurs nutritifs dans des cellules transformees Withdrawn EP1255841A2 (fr)

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DE10045182A DE10045182A1 (de) 2000-02-14 2000-09-13 Verwendung von Palatinase- und Trehalulase-Sequenzen als nutritive Marker in transformierten Zellen
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