EP0787196A1 - Dna-sequenzen und ihre verwendung - Google Patents

Dna-sequenzen und ihre verwendung

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Publication number
EP0787196A1
EP0787196A1 EP95938377A EP95938377A EP0787196A1 EP 0787196 A1 EP0787196 A1 EP 0787196A1 EP 95938377 A EP95938377 A EP 95938377A EP 95938377 A EP95938377 A EP 95938377A EP 0787196 A1 EP0787196 A1 EP 0787196A1
Authority
EP
European Patent Office
Prior art keywords
dna sequence
dna
plants
sequence
component
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP95938377A
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German (de)
English (en)
French (fr)
Inventor
Rüdiger Hain
Regina Fischer
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Bayer CropScience AG
Original Assignee
Bayer AG
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Filing date
Publication date
Application filed by Bayer AG filed Critical Bayer AG
Publication of EP0787196A1 publication Critical patent/EP0787196A1/de
Withdrawn legal-status Critical Current

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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.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • the present invention relates to a new DNA sequence and its use for the transformation of vectors, host organisms and plants and for the production of new plants which are male-sterile and have a changed flower color.
  • male sterile plants play an important role in plant breeding, especially in hybrid breeding.
  • Various methods for the production of male-sterile plants have already become known, according to which, for example, targeted cell damage z. B. caused in the anthers, interventions in mitochondrial functions take place, with the help of antisense DNA sterilizing possibilities of action of chemicals are created or the chalcone synthesis is inhibited (cf. WO 90/08830, WO 90/08831, WO 89/10396, EP -A-0 329 308 and EP-A-0 335 451).
  • the methods available hitherto for the production of male-sterile plants do not lead to fully satisfactory results in many cases.
  • plants with a significantly increased sensitivity to fungal pests are often obtained, which makes their practical handling to a great extent difficult. There is therefore a strong need for further processes for the production of male-sterile plants which do not have these disadvantages.
  • DNA sequence I The new DNA sequence, which is called DNA sequence I below, has now been found, which consists of the following constituents, which is shown in FIG.
  • 3'-sequence are: a) a promoter which is heterologous to component b) and which is highly active in plants and / or which is anther- or tapetum-specific and is optionally preceded by a reinforcing element ("enhancer");
  • DNA sequence I also including the derived DNA sequences which still have the features which are essential for carrying out the invention.
  • plants which contain DNA sequence I in their genome are surprisingly male-sterile and moreover have a flower color which is different from the corresponding plants which do not contain DNA sequence I.
  • the present invention thus also relates to new plants (including parts of these plants and their propagation material, such as protoplasts, plant cells, calli, seeds, tubers or cuttings, etc.) which contain the DNA sequence I in their genome and which are male-sterile and / or a flower color which has changed compared to the corresponding plants which do not contain the DNA sequence I.
  • Promoters which can be used according to the invention as constituent a) of DNA sequence I and which are highly active in plants are known.
  • the promoter of the gene of the small subunit of ribulose-1, 5-bisphosphate-carboxylase (rbcS) can be mentioned as an example (see, for example, EMBO Journal, Vol. 5 No. 9, 2063-2071 (1986)).
  • highly effective plant virus promoters can also be used in plants.
  • Such promoters are known, the CaMV 35S promoter (cf. for example Science 250: 959-960 (1990) being mentioned as an example.
  • Anther-specific and or wallpaper-specific promoters can also be used as component a) of the DNA sequence I.
  • Promoters of this type which are particularly effective in the anthers or in the anther site called tapetum are known.
  • the TA29 promoter should be mentioned as an example (cf., for example, Nature 347, 737-741 (1990)).
  • the known anther-specific promoters of the TA26 and TA13 genes isolated from tobacco are also suitable for use according to the invention.
  • the CaMV 35S promoter is preferably used as component a) of the DNA sequence I.
  • enhancer-promoter constructs are known.
  • the known CaMV 35S enhancer can be used particularly advantageously as an enhancer.
  • the CaMV 35S promoter is particularly preferably used as component a) of the DNA sequence I.
  • a construct is very particularly preferably used which consists of the CaMV 35S enhancer and the CaMV 35S promoter which follows in 5'-3 'sequence.
  • the promoter to be used according to the invention is heterologous to component b), i.e. different from promoters which occur in natural stilbene synthase genes.
  • Stilbene synthase should be understood to mean any enzyme which (in a suitable environment, especially in plant cells) can produce stilbene.
  • the term stilbene describes a group of chemical substances that occur in plants and contain the stilbene framework (trans-1,2-diphenylethylene) as a common basic structure. This basic structure can also be supplemented by adding further groups.
  • Two important and preferred stilbenes are the 3,5-dihydroxy-stilbene (Pinosylvin) and the 3,4 ', 5-trihydroxy-stilbene (resveratrol).
  • DNA sequences encoding stilbene synthase are known, for example, from European patent applications EP-A-0 309 862, EP-A-0 464 461 and EP-A-0 533 010. These patent applications describe the isolation of stilbene synthase genes and their use for the production of transgenic plants which have increased pest resistance.
  • the DNA sequences coding for stilbene synthase which are described in these patent applications, are preferably used according to the invention, the sequences coding for resveratrol synthase being particularly preferred.
  • the DNA sequences from peanut plants which are described in the European patent applications and which code for stilbene synthase are preferably used.
  • the DNA sequences coding for stilbene synthase can be in the form as they are contained in the corresponding natural plant genes ("genomic form"), including any non-coding regions (such as introns), or in a form which the corresponds to cDNA ("copy DNA”), which is obtainable via mRNA with the help of reverse transcriptase / polymerase and no longer contains introns. They can also be in partially or completely synthesized form or be composed of parts from different origins.
  • Particularly preferred according to the invention are the DNA sequences coding for stilbene synthase, which are contained in the plasmid pGS828.1 (EP-A-0 309 862), the plasmid pin5-49 (EP-A-0 533 010) and very particularly preferably the plasmids pVstl , pVst2 and pVstl2t3 (EP-A-0 464 461) are contained and the further DNA sequences coding for stilbene synthase which can be isolated from plants with the aid of these DNA sequences (use as probes) are used.
  • the sequence coding for stilbene synthase, which is contained in the plasmid pVstl (EP-A-0 464 461) is particularly emphasized.
  • the isolation of the DNA sequences which can be used as component b) of the DNA sequence I is known and / or can be carried out by the known methods and methods familiar to the person skilled in the art.
  • the coding region for stilbene synthase can e.g. B. with the help of the polymerase chain reaction technique (PCR technology) from the plasmids pVstl, pVst2 pVstl2t3 or pGS828.1 isolated.
  • the amplification can be done by means of PCR, e.g. B. via the following programs:
  • Primer 1 Vstl see SEQ ID NO: 1 primer IVst2: see SEQ ID NO: 2 primer 1 A. hyp .: see SEQ ID NO: 3
  • Primer 2 Vstl see SEQ ID NO: 4
  • Primer 2 Vst2 see SEQ ID NO: 5
  • Primer 2 A. hyp . see SEQ ID NO: 6
  • the coding and the terminating sequence can also be isolated from pSSVstl (see below) by the enzymes EcoRI and PstI and EcoRI and SphI.
  • the 3'-polyadenylation sequence contained as component c) in the DNA sequence I can be varied to a large extent, so that all corresponding sequences can be used which do not adversely affect the expression of stilbene synthase in plants. It can also be expedient to use several (for example two) polyadenylation sequences, optionally of different origins, to be used one after the other, especially if this results from the techniques used (see part c) in SEQ ID NO: 7).
  • the 3'-polyadenylation sequence contained in the natural stigen synthase genes is preferably used, this sequence advantageously being isolated from the stilbene synthase genes together with the sequence coding for stilbene synthase.
  • stilbene synthase genes can therefore also be used as constituents b) and c) in which only the natural promoter has been removed.
  • component a) of DNA sequence I that is to say the heterologous promoter and, if appropriate, the enhancer.
  • Suitable 3'-polyadenylation sequences can be isolated by generally customary methods and methods which are familiar to the person skilled in the art.
  • nucleotides 1 to 720 represent the double 35S CaMV RNA promoter, which consists of the CaMV 35S enhancer and the CaMV 35S promoter (component a)).
  • Nucleotides 721 to 730 are a synthetic linker sequence.
  • Nucleotides 731 to 2265 of SEQ ID NO: 7 represent the coding part for stilbene synthase (component b)) and nucleotides 2266 to 2485 represent the polyA part (component c)) of the stilbene synthase gene.
  • the nucleotides from 2486 to 2728 represent the portion of component c) from CaMV 359 RNA, with polylinker sequences at the end.
  • DNA sequence I also includes all derived DNA sequences which still have the features which are essential for the implementation of the invention, which therefore cause male sterility in plants and, if appropriate, a change in the color of the flowers.
  • individual DNAs, codons and / or partial sequences may be missing (eg through the use of restriction enzymes) and / or replaced by other DNAs, codons and / or partial sequences.
  • modifications can be due to the degeneracy of the genetic code or can result from the manipulation of the DNA sequences.
  • the DNA sequences according to the invention and / or their components a) to c) can also contain DNAs and / or DNA sequences which facilitate their handling, for example so-called. Left or which of such links core remains after manipulations (eg after cuts with restriction enzymes).
  • the components a) to c) of the DNA sequence I can be of natural origin or can be partially or completely in synthesized form.
  • the DNA sequence I consists of (a) the so-called CaMV 35S double promoter, which is composed of the CaMV 35S promoter and the associated CaMV 35S enhancer, and (b) the sequence coding for stilbene synthase (resveratrol synthase) with the subsequent 3'-polyadenylation sequence, as is present in the plasmid pVstl (cf. EP-A-0 464 461).
  • This DNA sequence is contained in the new plasmid pSSVstl, the construction of which can be seen from FIG. 1.
  • the coding region of the stilbene synthase gene Vstl can accordingly be isolated as a 2.1 kB Munl fragment from the plasmid pVstl, which contains the complete stilbene synthase gene (Vstl gene) as a 4.9 kB EcoRI fragment.
  • this Muni fragment lacks the first 4 codons at the 5 'end of the coding area.
  • the purified Muni fragment is expediently digested with the restriction enzyme Nrul and the resulting 1.7 kB large Nrul Munl fragment is fused with an oligonucleotide linker which codes for the first four amino acids.
  • the oligonucleotide linker is designed in such a way that the EcoRI site is only created by a subsequent restriction digest.
  • the resulting NruI / EcoRI fragment is ligated between the Smal and EcoRI sites of the binary vector pSS, so that the complete coding region of the stilbene synthase gene Vstl is under the control of the double 35S promoter.
  • Corresponding other constructions can be produced and used by the person skilled in the art on the basis of his specialist knowledge and the information contained in the present text using the usual methods.
  • the Escherichia coli strain RH pSSVstl contains the plasmid pSSVstl.
  • This E. coli strain RH pSSVstl was obtained from the German Collection of Microorganisms (DSM), Mascheroder Weg 1B, D-38124 Braunschweig, Federal Republic of Germany, in accordance with the provisions of the Budapest Treaty on the International Filing of Microorganisms for the purposes of patent proceedings on October 18, 1994 and received the deposit number DSM 9501.
  • the plasmid pSSVstl and the E. coli strain RH pSSVstl and its mutants, which still have the essential features of the stored strain for the implementation of the invention, are also part of the present invention.
  • the E. coli strain RH pSSVstl can be multiplied by the generally customary methods.
  • the plasmid pSSVstl can also be obtained from this E. coli strain using the generally customary methods. It is also easily possible for the person skilled in the art to isolate the DNA sequence I contained in the plasmid pSSVstl. For example, the DNA sequence I contained in the plasmid pSSVstl is isolated from this plasmid with the aid of the restriction enzymes SphI and PstI in the form of an approximately 2700 bp (base pairs) large DNA fragment.
  • DNA sequence I one or more times (for example a tandem arrangement), preferably simply, into any prokaryotic (preferably bacterial) or eukaryotic (preferably vegetable) DNA as “foreign” DNA to install.
  • prokaryotic preferably bacterial
  • eukaryotic preferably vegetable
  • the "modified" recombinant DNA which e.g. can be used for the transformation of plants or plant cells and is contained in plants or plant cells after the transformation, is part of the present invention.
  • the DNA sequence I and the recombinant DNA can be used as "foreign" DNA in
  • Vectors in particular plasmids, cosmids or phages
  • transformed microorganisms preferably bacteria, in particular Gram-negative bacteria, such as E. coli
  • transformed plant cells and plants or in their DNA are constituents of the present invention.
  • the DNA sequence I is built into the natural plant genome one or more times (at the same or different locations in the genome).
  • the present invention thus also relates to a process for the production of transgenic plant cells (including protoplasts) and plants (including plant parts and seeds), these plants being male-sterile and possibly having a changed flower color, which is characterized in that
  • the DNA sequence I and / or recombinant DNA according to the invention is inserted one or more times into the genome of plant cells (including protoplasts) and, if appropriate
  • the desired plant parts are obtained from the transgenic plants of the parent generation or further generations obtained therefrom.
  • Process steps (a), (b) and (c) can be carried out in a conventional manner by known processes and methods.
  • Transgenic plant cells including protoplasts
  • plants including plant parts and seeds
  • Parts of the present invention are also:
  • transgenic plant cells according to the invention including protoplasts
  • plants including plant parts and seeds
  • transgenic plants which are male-sterile and / or have a different flower color compared to corresponding plants which do not contain this DNA in their genome.
  • a number of different methods are available for inserting the DNA sequence I as "foreign" DNA into the genetic material of plants or plant cells.
  • the gene transfer can take place in accordance with the generally known methods, the person skilled in the art being able to determine the most suitable method in each case without difficulty.
  • the Ti plasmid from Agrobacterium tumefaciens is available as a particularly inexpensive and widely usable vector for the transfer of foreign DNA into genomes of dicotyledonous and monocotyledonous plants.
  • the DNA sequence I is suitably inserted into the T-DNA of suitable Ti plasmids (for example Zambryski et al., 1983) and by infection of the plant, infection of parts of plants or plant tissues, such as, for example, leaf disks, stems , Hypocotyls, cotyledons, meristems and tissues derived therefrom, such as secondary embryos and calli or by co-culture of protoplasts with Agrobacterium tumefaciens.
  • suitable Ti plasmids for example Zambryski et al., 1983
  • DNA uptake can also be promoted by an electric field (electroporation) (e.g. Fromm et al., 1986).
  • electroporation e.g. Fromm et al., 1986.
  • the DNA can also be introduced via plant pollen in a known manner, e.g. by "bombarding" pollen or plant tissue with physically accelerated particles which carry the DNA (cf. EP-A 0 270 356).
  • the plants are regenerated in a known manner using suitable nutrient media (e.g. Nagy and Maliga 1976).
  • suitable nutrient media e.g. Nagy and Maliga 1976.
  • Plasmid pSSVstl is contained in a suitable intermediate E. coli vector e.g. pGV700 or pGV710, (see EP-A-116 718) or preferably derivatives thereof, which additionally contain a reporter gene such as e.g. nptll (Herrera-Estrella et al. 1983) or hpt (Van den Elzen et al 1986).
  • a reporter gene such as e.g. nptll (Herrera-Estrella et al. 1983) or hpt (Van den Elzen et al 1986).
  • the plasmid constructed in this way is transferred to Agrobacterium tumefaciens, which contains, for example, pGV 3850 or derivatives thereof (Zambryski et al. 1983), using customary methods (for example Van Haute et al. 1983).
  • the DNA sequence I can be cloned in a binary vector, for example PCV001 or PCV002 (for example Koncz and Schell 1986) and transferred to a suitable Agrobacterium strain (Koncz and Schell 1986) as described above.
  • the resulting Agrobacterium strain which contains the DNA sequence I in a form that can be transferred to plants, is subsequently used for plant transformation.
  • the plasmid pSSVstl can also be introduced directly into a suitable A.
  • the plasmid pSSVstl which contains a reporter gene for plant cells for kanamycin resistance (for example Herrera-Estrella et al. 1983), is transferred to plant protoplasts in a conventional manner (for example Hain et al 1985).
  • the plasmid pSSVstl can be present in a circular, but preferably in a linear form.
  • Transformed (transgenic) plants or plant cells are produced according to the known methods, e.g. by leaf disc transformation (e.g. Horsch et al. 1985) by coculturing regenerating plant protoplasts or cell cultures
  • Agrobacterium tumefaciens e.g. Marton et al. 1979, Hain et al. 1985
  • Resulting transformed plants are either selected by selection for the expression of the reporter gene, e.g. by the phosphorylation of kanamycin sulfate in vitro (Reiss et al. 1984; Schreier et al. 1985) or by the expression of nopaline synthase (according to Aerts et al. 1983) or
  • Stilbene synthase detected by Northern blot analysis and Western blot analysis.
  • the stilbene synthase and the stilbene can also be detected in a known manner with the aid of specific antibodies in transformed plants.
  • Stilbene synthase can also be detected by enzyme activity test (Rolfs et al., Plant Cell Reports J, 83-85, 1981).
  • the cultivation of the transformed plant cells and the regeneration to complete plants is carried out according to the generally customary methods with the aid of the appropriate nutrient media.
  • Both the transformed plant cells and the transformed plants which contain the DNA sequence I according to the invention and which are components of the present invention show a considerably higher resistance to pests, in particular phytopathogenic fungi.
  • plants means both complete plants and parts of plants, such as leaves, stems or roots, and propagation material, such as seeds, tubers, cuttings, etc.
  • Plant cells include protoplasts, cell lines, plant calli, etc.
  • the flower color is of considerable commercial importance.
  • the targeted influencing of the flower colors and the achievement of stable flower colors is often difficult and complex.
  • the present invention makes it possible to change the flower color of all brightly flowering plants which have flower colors, in particular anthocyanins, in a relatively simple manner.
  • the incorporation of DNA sequence I makes the flowers lighter and often completely white. In plants without colored flowers, a change is generally not or only with difficulty.
  • the male sterility of plants plays a very important role in plant breeding in the production of hybrid lines and hybrid seeds.
  • many hybrid lines are very sensitive to phytopathogenic fungi, so that their use is very limited.
  • With the help of the present invention it is possible to produce male-sterile plants in a relatively simple manner. These plants also have an increased resistance to microbial plant pests, such as phytopathogenic fungi, bacteria and / or viruses, in particular to phytopathogenic fungi, and are therefore superior to male-sterile plants obtained by other processes.
  • the plants which can be given male sterility by incorporating (transforming) the DNA sequence I according to the invention include practically all plants.
  • crops such as plants that supply food and raw materials, e.g. cereals (in particular wheat, rye, barley, oats, millet, rice and corn), potatoes, legumes (such as legumes and in particular alfalfa, soybeans), vegetables (especially types of cabbage and tomatoes), fruit (especially apples, pears, cherries, grapes, citrus, pineapples and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal and cotton, as well as medicinal plants such as rauwolfia and digitalis.
  • Rice, wheat, barley, rye, corn, sugar beet, rapeseed and soy are particularly preferred. The present invention will be explained in more detail using the following exemplary embodiments:
  • the plasmid pSSVstl is a derivative of pSS.
  • pSS is a derivative of PCV001 (Koncz and Schell, 1986), which contains an expression cassette based on the plasmid pRTlOl (Töpfer et al., 1987), in which a doubling of the CaMV 35S RNA enhancer by cloning the Ddel / EcoRV- Fragment in the Hincll interface was carried out.
  • pSSVstl contains the coding sequence and the polyA sequence of stilbene synthase from pVstl (cf. FIG. 1).
  • pSSVstl contains a kanamycin resistance for plants and a bacterial ampicillin resistance.
  • pSSVstl contains border sequences from the Ti plasmid from Agrobacterium tumefaciens and a start of replication for A. tumefaciens and E. coli
  • the plasmid pSSVstl can be mobilized using the E. coli RH pSSVstl strain directly into a suitable Agrobacterium tumefaciens strain (e.g. Koncz and Schell, 1986).
  • Nicotiana tabacum (Petit Havana SRI) is propagated as a sterile shoot culture on hormone-free LS medium (Linsmai er and Skoog 1965). At intervals of about 6-8 weeks, shoot sections are transferred to fresh LS medium. The shoot cultures are kept in a culture room at 24-26 ° C. under 12 h light (1000-3000 lux).
  • the leaf material is in 20 ml enzyme solution, consisting of K3 medium (Nagy and Maliga 1976), 0.4 m sucrose, pH 5.6, 2% cellulase RIO (Serva), 0.5% Macerozym RIO (Serva) incubated for 14-16 h at room temperature.
  • K3 medium Nagy and Maliga 1976
  • Serva cellulase RIO
  • Macerozym RIO Serva
  • the protoplasts are then separated from cell residues by filtration through 0.30 mm and 0.1 mm steel sieves.
  • the filtrate is centrifuged at 100 xg for 10 minutes. During this centrifugation, intact protoplasts float and collect in a band at the top of the enzyme solution.
  • the pellet from cell residues and the enzyme solution are aspirated with a glass capillary.
  • the pre-cleaned protoplasts are made up to 10 ml with fresh K3 medium (0.4 M 10 sucrose as an osmotic agent) and
  • the washing medium is suctioned off and the protoplasts are diluted to 1-2 ⁇ 10 5 / ml for culture or subsequent infection with agrobacteria (coculture).
  • the protoplast concentration is determined in a counting chamber.
  • Sequence I contained in their T-DNA or which contain the plasmid pSSVstl, added to 3 ml of regenerating protoplasts in minimally A (Am) medium (density approx. 10 9 agrobacteria / ml).
  • Am minimally A
  • the coculture lasts 3-4 days at 20 ° C in the dark.
  • the tobacco cells are filled into 12 ml centrifuge tubes with sea water
  • the cell suspension is at a density of 5 ⁇ 10 4 / ml in K3 medium (0.3 m saccharose) with 1 mg / 1 NAA (naphthyl-1-acetic acid), 0.2 mg / 1 kinetin
  • Approx. 10 6 protoplasts in 180 ⁇ l K3 medium with 20 ⁇ l aqueous DNA solution containing 0.5 ⁇ g / ⁇ l plasmid pSSVstl or the isolated DNA sequence I from pSSVstl as a DNA fragment and 0.5 ⁇ l / ⁇ l pLGVneo2103 are placed in a Petri dish (Hain et al. 1985), mixed carefully. Then 200 ⁇ l of fusion solution (0.1 m calcium nitrate, 0.45 M mannitol, 25% polyethylene glycol (PEG 6000), pH 9) are carefully added.
  • fusion solution 0.1 m calcium nitrate, 0.45 M mannitol, 25% polyethylene glycol (PEG 6000), pH 9
  • the transformation with the DNA sequence I from pSSVstl can also be carried out without the addition of the 0.5 ⁇ g / ⁇ l pLGV neo 2103. Since no reporter gene is used in this case, the resulting calli are checked for the presence of the DNA sequence I gene unit using a dot blot hybridization.
  • Hybridization sample the coding sequence from pSSVstl can be used.
  • detection methods such as a test with antibodies or an enzyme test for stilbene synthase, can also be used.
  • a modified "bead type culture” technique (Shillito et al. 1983) is used for the culture and selection of kanamycin-resistant colonies described below.
  • K3 medium 0.3 M sucrose + hormones; 1.2% (Seaplaque) LMT agarose (low melting agarose, Marine Colloids) mixed in 5 cm petri dishes for this purpose
  • the exchange medium (K3 + Km) is reduced by 0.05 m of sucrose (approx. 60 mOsm) per week.
  • kanamycin-resistant colonies have reached a diameter of approx. 0.5 cm
  • half is placed on regeneration medium (LS medium, 2% sucrose, 0.5 mg / 1 benzylaminopurine BAP) and exposed to 12 h light (3000-5000 lux ) and 24 ° C in the culture room.
  • the other half is propagated as callus culture on LS medium with 1 mg / 1 NAA, 0.2 mg / 1 kinetin, 0.1 mg / 1 BAP and 100 mg / 1 kanamycin sulfate.
  • the regenerated shoots are approx. 1 cm in size, they are cut off and placed on 1/2 LS medium (1% sucrose, 0.8% agar) without growth regulators for rooting.
  • the shoots are rooted on 1/2 MS medium with 100 mg / 1 kanamycin sulfate and later converted into soil.
  • Leaf pieces are then washed in MS medium (0.5 mg / ml BAP, 0.1 mg / ml NAA) and on the same medium (0.8% agar) with 500 g / ml cefotaxime and 100 g / ml kanamycin sulfate ( Sigma). The medium should be replaced after two weeks. Transformed shoots become visible after another 2-3 weeks.
  • MS medium 0.5 mg / ml BAP, 0.1 mg / ml NAA
  • Neomycin phosphotransferase (NPT II) enzyme test :
  • NPT II activity in plant tissue is determined by in situ phosphorylation of kanamycin, as described by Reiss et al. (1984) and by Schreier et al. (1985) modified as follows. 50 mg of plant tissue are homogenized in 50 ⁇ l extraction buffer (10% glycerol, 5% 2-mercaptoethanol, 0.1% SDS, 0.025% bromophenol blue, 62.5 mM Tris pH 6.8) with the addition of glass powder on ice and for 10 minutes centrifuged in an Eppendorf centrifuge at 4 ° C.
  • the gel is placed on a glass plate of the same size and covered with 40 ml of 1% agarose in reaction buffer which contains the substrates kanamycin sulfate (20 g / ml) and 20-200 Ci 32 P ATP (Amersham).
  • the sandwich gel is incubated for 30 min at room temperature and then a sheet of phosphocellulose paper P81 (Whatinan) is placed on the agarose.
  • P81 phosphocellulose paper
  • 3MM Whatinan
  • transgenic tobacco plants obtained according to the above examples are grown in tissue culture and then in the greenhouse at 23 ° C. and 70-
  • T L -DNA gene 5 controls the tissue- specific expression of chimaeric genes carried by a noval type of Agrobacterium linary vector. Mol. Gen. Genet. (1986) 204: 338-396
  • EP-A-174 166 WO 83/01176
  • EP-A-122 791 EP-A-0 309
  • EP-A-0 464 461
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CTCTCTACAA ATCTATCTCTCT CTCTATTTTT CTCCAGAATA ATGTGTGAGT AGTTCCCAGA 258

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EP95938377A 1994-11-10 1995-10-30 Dna-sequenzen und ihre verwendung Withdrawn EP0787196A1 (de)

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DE4440200 1994-11-10
DE4440200A DE4440200A1 (de) 1994-11-10 1994-11-10 DNA-Sequenzen und ihre Verwendung
PCT/EP1995/004256 WO1996015251A1 (de) 1994-11-10 1995-10-30 Dna-sequenzen und ihre verwendung

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US5955361A (en) * 1996-11-20 1999-09-21 Pioneer Hi-Bred International, Inc. P gene promoter constructs for floral-tissue preferred gene expression
FR2775001B1 (fr) * 1998-02-13 2000-05-12 Lvmh Rech Acide nucleique comprenant la sequence d'un promoteur inductible par un stress et une sequence d'un gene codant pour une stilbene synthase, cellule et plante transformees par cet acide nucleique
SG96587A1 (en) * 2000-11-21 2003-06-16 Nat Inst Of Education Nanyang Production of stilbenes in transgenic plants and the method of producing thereof
US7977049B2 (en) * 2002-08-09 2011-07-12 President And Fellows Of Harvard College Methods and compositions for extending the life span and increasing the stress resistance of cells and organisms
US20060025337A1 (en) * 2003-07-01 2006-02-02 President And Fellows Of Harvard College Sirtuin related therapeutics and diagnostics for neurodegenerative diseases
EP2236131A3 (en) * 2003-07-01 2011-03-02 President and Fellows of Harvard College Sirt1 modulators for manipulating cell/organism lifespan/stress response
US20050171027A1 (en) 2003-12-29 2005-08-04 President And Fellows Of Harvard College Compositions for treating or preventing obesity and insulin resistance disorders
US8017634B2 (en) * 2003-12-29 2011-09-13 President And Fellows Of Harvard College Compositions for treating obesity and insulin resistance disorders
WO2006007411A2 (en) * 2004-06-16 2006-01-19 President And Fellows Of Harvard College Methods and compositions for modulating bax-mediated apoptosis
US20060014705A1 (en) * 2004-06-30 2006-01-19 Howitz Konrad T Compositions and methods for selectively activating human sirtuins
CN100405061C (zh) * 2005-04-01 2008-07-23 石河子大学 梨树病毒杂交检测试剂盒及其检测方法
WO2006138418A2 (en) * 2005-06-14 2006-12-28 President And Fellows Of Harvard College Improvement of cognitive performance with sirtuin activators
CN115141838A (zh) * 2022-06-07 2022-10-04 珠海科技学院 一种白藜芦醇合酶基因转化花生体系的构建

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NL8800756A (nl) * 1988-03-25 1989-10-16 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Genetisch gemanipuleerde plantecellen en planten, alsmede daarvoor bruikbaar recombinant dna.
DE4107396A1 (de) * 1990-06-29 1992-01-02 Bayer Ag Stilbensynthase-gene aus weinrebe
HU219543B (hu) * 1991-04-16 2001-05-28 Mogen International N.V. Eljárás hímsteril növények és az eljárásban használható rekombináns DNS előállítására
DE4117747A1 (de) * 1991-05-30 1992-12-03 Bayer Ag Kaffeoyl-coa 3-o-methyltransferase-gene
DE4130986A1 (de) * 1991-09-18 1993-03-25 Bayer Ag Pinosylvinsynthase-gene
JP3418396B2 (ja) * 1992-03-09 2003-06-23 ワシントン ステート ユニバーシティ リサーチ ファンデーション 植物稔性の調節方法
AU6164794A (en) * 1993-01-29 1994-08-29 Monsanto Company Method of controlling plant pathogenic fungi

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JPH10508495A (ja) 1998-08-25
CN1117867C (zh) 2003-08-13
MX9703431A (es) 1997-07-31
HUT77108A (hu) 1998-03-02
AU3979395A (en) 1996-06-06
NZ296081A (en) 1998-07-28
CN1162979A (zh) 1997-10-22
BR9509641A (pt) 1997-09-16
WO1996015251A1 (de) 1996-05-23
HU222268B1 (hu) 2003-05-28
DE4440200A1 (de) 1996-05-15
AU700022B2 (en) 1998-12-17
US6063988A (en) 2000-05-16

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