WO2002066661A1 - Procede pour realiser et transformer des agglomerations de mitochondries - Google Patents

Procede pour realiser et transformer des agglomerations de mitochondries Download PDF

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WO2002066661A1
WO2002066661A1 PCT/EP2002/001737 EP0201737W WO02066661A1 WO 2002066661 A1 WO2002066661 A1 WO 2002066661A1 EP 0201737 W EP0201737 W EP 0201737W WO 02066661 A1 WO02066661 A1 WO 02066661A1
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mitochondrial
mitochondria
accession
protein
transformation
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PCT/EP2002/001737
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German (de)
English (en)
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Jens-Otto Giese
Uwe Sonnewald
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Ipk - Institut Für Pflanzengenetik Und Kulturpflanzenforschung
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Publication of WO2002066661A1 publication Critical patent/WO2002066661A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation

Definitions

  • the invention relates to a method for producing mitochondrial conglomerates to facilitate the subsequent transformation of the mitochondria, wherein the production of the mitochondrial conglomerates involves the expression of a first protein component which is capable of being anchored in the outer mitochondrial membrane and a second protein component capable of sterically shielding determinants on the surface of mitochondria in which the cells containing mitochondria are comprised.
  • Mitochondria like plastids, are organelles from eukaryotic cells. Both the mitochondria and the plastids, especially chloroplasts, contain their own DNA. Generally are
  • mitochondria and chloroplasts divide by constriction.
  • a ring of tubulin-related proteins is formed, which constricts with GTP consumption.
  • this ring consists of FtsZ.
  • FtsZ FtsZ.
  • no ftsz homologues were found that could be responsible for mitochondrial division, but in the unicellular algae Mallomonas splendens and the unicellular red algae Cyanidioschyzon merolae.
  • yeast and animals the mitochondria are constricted with the participation by Dynamin. Dynamins were previously known to be associated with endocytosis, where they lead to constriction of the endocytosis vesicles.
  • the mitochondrion is surrounded by two highly specialized membranes, the outer membrane and the inner membrane, which are crucial for its activity.
  • Each of the two lipid bilayers contains a unique protein pattern, and together they enclose and define two separate mitochondrial compartments: the inner matrix space and the narrow space between the two membrane systems (intermembrane space).
  • mitochondria would also offer the advantage that they are also productive in large quantities in seeds.
  • transformation processes that are used for the transfer of Foreign genes on mitochondria are suitable, for example micromanipulation or bombardment with DNA.
  • a (fusion) protein which comprises two components, namely a first, which causes the protein to be anchored in the outer mitochondrial membrane, and a second, which is able to determine determinants on the Sterically shielding the surface of mitochondria, which causes the formation of mitochondrial conglomerates.
  • the phenomenon now observed for the first time is not a random event, but a reproducible way of providing larger mitochondrial structures that can be genetically manipulated using conventional transformation methods due to their size.
  • Manufacturing shielding component is described here for the first time.
  • Mitochondrial lumps were developed by Rubino et al. (2001, Journal of General Virology 82: 29-34) after transient expression of the 36K viral protein, fused to GFP, described in Nicotiana benthamicana, but the size changes referred to there as "dumping" are not genetically manipulable conglomerates in the The meaning of the present invention by Rubino et al. (2001, vide supra) EM recordings only show that individual mitochondria get bigger, change their structure and can clump together, with a maximum of two clumped mitochondria being shown.
  • the method according to the invention effects a conglomeration of several (usually at least 3 or more) mitochondria, as is necessary for the use of the mitochondria for genetic manipulation.
  • CIRV Carnation Italian Ringspot Virus
  • MVBs multivesicular bodies
  • the fusion proteins used in the context of the invention do not destroy the mitochondrial membrane.
  • the transgenic plants in which the mitochondrial conglomeration takes place preferably do not show any visible phenotype. Without wishing to be bound by any hypothesis, it is currently assumed that the expression of the protein components according to the invention inhibits mitochondrial division, which results in mitochondrial conglomerates which could be termed “giant mitochondria” and which can be genetically manipulated due to their size .
  • the ("giants") mitochondria produced according to the invention are significantly larger than the naturally occurring mitochondria and consist of several mitochondria.
  • the mitochondria according to the invention preferably have a size of at least 2 ⁇ m, 3 ⁇ m, more preferably at least 4 ⁇ m, 6 ⁇ m and particularly preferably of at least 7 ⁇ m, 8 ⁇ m Most preferably, the mitochondria have a size of at least 9 ⁇ m, 10 ⁇ m or more in diameter, and the particularly preferred mitochondria are therefore comparable in size to the cell nucleus.
  • the anchoring component is the enzyme hexokinase (HK) or parts of this enzyme which are capable of anchoring in the outer mitochondrial membrane.
  • HK hexokinase
  • Other preferred anchoring components are porins or parts thereof which are capable of anchoring in the outer mitochondrial membrane.
  • any protein or part of a protein is suitable which has a cytoplasmic localization with anchoring in the outer mitochondrial membrane.
  • Hxk 1 from Nicotiana tabacum, for example, is an anchoring component.
  • the sequence of Hxk 1 was published under accession no.
  • Hxk 1 is a chloroplast protein.
  • the sequence of the hexokinase from tobacco was obtained by using a previously characterized hexokinase from spinach (Accession No. AF 118132) a cDNA library from tobacco was screened (Wiese et al. (1999) FEBS Lett. 461: 13-18).
  • the tobacco hexokinase obtained was obviously not subsequently characterized, but only the description of the spinach hexokinase was taken over, which indicates a localization of the hexokinase I in the outer envelope membrane.
  • hexokinases can also be used in the context of the invention, sequences suitable for creating suitable expression vectors can be found in the gene database, for example, under the keyword “hexokinase” (for example, at the filing date, there were over 1300 nucleic acid sequences for hexokinases recorded in the NCBI library) partially overlap).
  • Anchorage domain can be used, known.
  • Various proteins that are anchored in the outer mitochondrial membrane are listed below. The person skilled in the art can, using the references below, use conventional cloning methods to prepare suitable fusion proteins and use them to generate the desired mitochondrial conglomerates.
  • MMM1_YEAST Accession No. P41800: Burgess S.M., Delannoy M., Jensen R.E. "MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria.”
  • MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19.
  • a yeast mitochondrial outer membrane protein essential for protein import and cell viability is essential for protein import and cell viability.
  • the person skilled in the art can also use the proteins described in the prior art, which have a cytoplasmic localization with anchoring in the outer mitochondrial membrane, in particular hexokinases and porins, to find other suitable proteins, for example by PCR using oligonucleotides that are of known nucleic acid sequences are derived, or by hybridization of cDNA or genomic libraries with gene probes which correspond to known nucleic acid sequences.
  • the second component of the protein, the expression of which in plant cells leads to the desired formation of mitochondrial conglomerates, is the so-called shielding component, which causes steric shielding of determinants on the mitochondrial surface.
  • GFP green fluorescent protein
  • Shielding component should have a size that corresponds to a molecular weight of about 20 kDa, preferably 25 kDa and particularly preferably at least 28 kDa.
  • the anchoring component and the steric components are preferably expressed in the form of a fusion protein in the plant cell, i.e. the fusion protein is encoded by a chimeric gene construct that is an artificial one
  • the promoter controlling the expression of the fusion protein is a constitutive promoter, such as the 35S RNA promoter from CaMV, or a leaf-specific promoter.
  • a large number of cloning vectors are available to prepare for the introduction of foreign genes into higher plants or their cells Replication signal for E. coli and a marker gene for the selection of transformed bacterial cells included.
  • examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc.
  • the desired sequence in this case the fusion of (i) sequences which code for the anchoring component with (ii) sequences which are for the steric Coding shielding component can be introduced into the vector at a suitable restriction interface.
  • the plasmid obtained is then used for the transformation of E. cob ' cells.
  • Transformed E. cob ' cells are grown in an appropriate medium and then harvested and lysed, and the plasmid is recovered.
  • plasmid DNA is used for restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences.
  • a large number of techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method in each case without difficulty. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation medium, the fusion of
  • Proplastics injection, electroporation, direct gene transfer of isolated DNA into protoplasts, the introduction of DNA using the biolistic method and other options that have been well established for several years and are part of the usual repertoire of the specialist in plant molecular biology or plant biotechnology .
  • plasmids When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies for direct gene transfer. Simple plasmids such as e.g. B. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is recommended.
  • the usual selection markers are known to the person skilled in the art and it is not a problem for him to select a suitable marker.
  • DNA sequences may be required.
  • z. B. for the transformation of 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 with the genes to be introduced become.
  • 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.
  • Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation).
  • Binary vectors can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria.
  • the agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present.
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants can then be regenerated from the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells.
  • Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Whole plants can then be regenerated from the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells.
  • the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker which shows the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin and the like. a. mediated.
  • the individually selected marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA.
  • Alternative markers are also suitable for this, such as nutritive markers and screening markers.
  • selection markers can also be completely dispensed with, but this is associated with a fairly high need for screening. If marker-free transgenic plants are desired, strategies are available to the person skilled in the art which permit subsequent removal of the marker gene, e.g. B. cotransformation, sequence-specific recombinases.
  • the regeneration of the transgenic plants from transgenic plant cells is carried out according to customary regeneration methods using known nutrient media.
  • the plants thus obtained can then be prepared using conventional methods, including molecular biological methods, such as PCR, blot analyzes, for the presence of the introduced nucleic acid, which codes for the fusion protein, consisting of anchoring component and shielding component, are examined.
  • the transgenic plant or the transgenic plant cells can be any monocot or dicot plant or its plant cell. They are preferably useful plants or cells of useful plants. It is particularly preferably corn, rice, wheat, barley, oats, rye, soybean, rapeseed, potato, tomato, tobacco, sugar beet, pea, banana, pineapple, paprika, yams, cassava, cotton.
  • the plant cells transformed with the fusion gene construct coding for the fusion protein show the inventive phenomenon of the formation of mitochondrial conglomerates, due to an inhibition of the naturally occurring mitochondrial division. This inhibition is mediated by anchoring the fusion protein in the outer mitochondrial membrane, caused by the anchoring component of the fusion protein, and shielding determinants on the mitochondrial surface, caused by the steric component of the fusion protein.
  • the mitochondrial conglomerates of the transformed plant cells can be genetically modified due to their size using known transformation methods.
  • Particle bombardment or microinjection is particularly suitable as a transformation method, but other methods are also conceivable if they are suitable for the transformation of plastids or protoplasts, for example, or have proven them for these objects.
  • the invention thus also relates to plant cells and plants which have mitochondria which have been transformed by the method according to the invention, and to propagation material and harvest products of these plant cells or plants, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings, etc.
  • 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. cob ' cells, cultivation of bacteria, recombinant sequence analysis DNA, were according to Sambrook et al. (1989, vide supra).
  • E. coli (XL-1 Blue) bacteria were purchased from Stratagene (La Jolla, California, USA).
  • the Agrobacterium strain used for the plant transformation (C58C1 with the plasmid pGV 3850kan) was developed by Debleare et al. (1985, Nucl. Acids Res. 13, 4777).
  • the vectors pCR-Blunt (Invitrogen, Carlsbad, California, USA), pBluescript SK- (Stratagene) and pBinAR (Höfgen and Willmitze (1990) Plant Sei. 66: 221-230) were used for the cloning.
  • the leaf disks were washed on MS medium with 100 mg / 1 kanamycin, 500 mg / 1 claforan, 1 mg / 1 benzylaminopurine (BAP), 0.2 mg / 1 naphthylacetic acid ( NAA), 1.6% glucose and 0.85 Bacto agar and the cultivation (16 h light / 8 h dark) continued.
  • Growing sprouts were transferred to hormone-free MS medium with 2% sucrose, 215 mg / 1 claforan and 0.8% Bacto agar.
  • Hxkl Two fusion constructs of hexokinase 1 (Hxkl, see above) were generated with the Green Fluorescent Protein (GFP), in which the Hxkl content was different.
  • the Hxkl and GFP fusion constructs are shown in Fig. 1.
  • HxklN GFP (N stands for N-terminus)
  • N stands for N-terminus
  • Hxkl the entire sequence coding for Hxkl was combined with that for GFP.
  • PCR Polymerase chain reaction
  • HK9GFP3 GAA TTC GGA CTT ATC TTC AAG
  • GTA HK9GFP5 GGA TCC C AA CTT TTA GCC AAC CTC C
  • HK9LGFP3 GAA TTC ACG AAG AAT AGC CAT AGC
  • HKI cDNA clone was used as a template for Hxkl and was isolated from a cDNA library which is identical to the one from which the one described by Wiese et al. published clone Hxkl was isolated (Wiese et al. (1999) FEBS Lett. 461: 13-8.).
  • the vector pBin-mGFP5-ER was used as the template for GFP (Siemering K.R. et al. (1996), Curr. Biol. 6: 1653-1663).
  • composition of the PCR reaction batches and the PCR program used in each case are given below.
  • nucleic acid sequences of the fusion constructs described above are given below, along with the amino acid sequences of the fusion proteins encoded by the fusion constructs.
  • Nucleic acid sequence of the HxklN : GFP fusion construct
  • the first 25 bases correspond to the sequence of the PCR primer HK9GFP5, including the BamHI restriction site (italic).
  • the Hxkl ATG start codon is highlighted.
  • the EcoRI restriction interface, via which the GFP fragment was connected to the Hxkl fragment, is also shown in italics.
  • the last 29 bases result from the PCR primer K76 used (counter strand, including Sall restriction site).
  • the EcoRI fusion results in amino acids 43-44, EF.
  • the EcoRI interface GAATTC is already in the GFP template pBin-mGFP5-ER, as a result of which an endoplasmic reticulum signal sequence (pBin-mGFP5-ER) was connected upstream.
  • this signal sequence was not used in the context of the invention and the EcoRI nucleotides are not necessary for the function of GFP (cf. access numbers U87974 with and U87973 without this restriction site).
  • the sequence of Hxkl at the relevant point is GAATTT, which is also translated to EF.
  • the amino acids EF can therefore still be included in the hexokinase sequence.
  • Hxkl GFP the Hxk stop codon TAG was mutated by EcoRI in GAA.
  • the amino acids EF are located at positions 498-499.
  • Epidermis was peeled off from the underside of a sheet and irradiated with light of the wavelengths 488 and 543 nm on the CLSM 410 from Zeiss (Jena, Germany). The emissions were limited to 510 to 525 nm by a bandpass filter. GFP protein became visible in light green.
  • the fusion constructs were transferred to tobacco cells as indicated above and transgenic plants were regenerated. Epidermis samples of the transgenic plants showed green glowing signals of the GFP in the fluorescence microscope. What was surprising at first, however, was that the two constructs had different distribution patterns from GFP showed. While the small construct caused several small glowing GFP signals, the larger construct marked significantly larger glowing areas, comparable in size to the cell nucleus. The electron microscopic examination of the plant tissue then showed that the large GFP-marked areas were accumulations of mitochondria that were aggregated to varying degrees. Peroxisomes were also associated with these aggregates. In the plants transformed with the smaller construct, HxklN:: GFP, the mitochondria also showed an affinity for one another, but to a lesser extent than with Hxkl :: GFP.
  • Oligomycin specifically inhibits the mitochondrial FoFj ATPase (hence the name FO for oligomycin-sensitive; this name was then retained for the chloroplastid ATPase, although it is not sensitive).
  • FO mitochondrial FoFj ATPase
  • 6 mutations were found in the gene for the subunit, which are probably for the
  • the first step towards a reproducible protocol is a mutated one
  • the second step is the production of a gene which contains a) oligomycin resistance as a marker and b) the biotechnologically or physiologically interesting gene under the control of a mitochondrial promoter (for the mitochondrial genome of Arabidopsis thaliana see, for example, Unseld et al. (1997) Nat. Genet. 15: 57-61).
  • the chimeric gene from oligomycin-resistant ATPase subunit, promoter and transgene is provided with mitochondrial DNA at both ends, which enables it to be homologously recombined into the target genome. Larger areas of the target genome may be exchanged. Here, the expert has to decide which areas of the original mitochondrial genome are unnecessary.
  • the original gene for ATPase subunit is replaced by the marker gene. Important genes may be replaced by the transgene that also integrates at this point.
  • the chimeric gene integrates at an unimportant location in the mitochondrial genome, which ensures the survival of the original genes.
  • the marker gene for oligomycin resistance does not replace the original gene for the ATPase subunit, but this also remains.
  • the transformed mitochondrial genome would have two genes for the ATPase subunit, with only one conferring oligomycin resistance. The disadvantage here would be reduced resistance.
  • GUS ß-glucuronidase
  • transgenic tobacco plants were also produced which expressed a fusion protein consisting of Hxkl and GUS ( ⁇ -glucuronidase).
  • the plasmid pCambia 1304 (Cambia, Canberra) was amplified from the plasmid GUS and NOS terminator and ligated into the vector pBinAR via Sall / Hindlll (the sequences for GUS and NOS, or another terminator, of course also from other common
  • the OCS terminator of the BinAR vector was replaced by the NOS termination sequences of the Cambia plasmid.
  • the vector's multiple cloning site has been expanded to include a Spel interface.
  • the new vector created in this way was named pGUS-AR.
  • Hxkl was amplified from the corresponding cDNA using the oligonucleotides HK9GFP5 and HK9-GFP-GUS_3 and ligated into the new vector pGUS-AR via BamHI / Spei.
  • the PCR was carried out under standard conditions using the following primers:
  • the Hxkl :: GUS construct was introduced into tobacco plants of the SNN variety by means of Agrobacterium-mediated transformation. Selection was carried out on medium containing kanamycin. Resistant plants were screened using qualitative GUS staining (see e.g. Jefferson (1987) Plant Mol. Biol. Rep. 5: 387-405; Jefferson et al. (1987) EMBO J. 6: 3901-3907). 21 GUS-positive plants were identified and analyzed for the presence of mitochondrial conglomerates.
  • Arabidopsis thaliana was also transformed with expression vectors which code for fusion proteins according to the invention.
  • the desired mitochondrial conglomeration was also observed in transgenic Arabidopsis pu zes.
  • transgenic tobacco and the transgenic Arabidops w plants show no visible phenotype.
  • SHAM Unlike by Ortega et al. described, SHAM also had an impact on callus growth alone. A clear effect was seen at more than 100 ⁇ M. At AA and Myx were already visible at 50 ⁇ M concentration inhibition, whereby Myx was somewhat stronger.
  • the concentration of SHAM should not exceed 100 ⁇ M at least in the initial stage.
  • the concentrations of AA and Myx should be at least 50 ⁇ M.
  • oligonucleotides were derived, with the aid of which the complete ORF of the cob gene was amplified from tobacco DNA. After ligation into the vector pCR blunt (Invitrogen), those mutations were introduced by means of PCR which should lead to resistance to AA or Myx.
  • the mutation of nucleotide gl28 to t leads to the amino acid exchange glycine-43 ⁇ valine.
  • Mutation of nucleotide t405 to a leads to the amino acid exchange phenylalanine-135 -> leucine.
  • nucleotide t423 was mutated to a in both plasmids, creating an Ncol cleavage site without changing the amino acid sequence.
  • the resulting plasmids were cobG43V + NcoI illustration. called cobF135L + NcoI.
  • the construction of the plasmids used for the selection of transgenic mitochondria is shown in Figure 5.
  • Tobacco plants of the Havana variety were transformed by means of Agrobacterium-mediated gene transfer with Hxkl :: GFP.
  • the positive plants selected on kanamycin showed the well-known phenomenon of large mitochondrial conglomerates.
  • the Havana variety was used because it has proven itself in plastid transformation.
  • Leaf discs were taken from the transgenic tobacco plants with mitochondrial conglomerates, as well as from wild types. Using a particle gun, these were washed one to three times with the plasmid cobG43V + NcoI entitled bombarded cobF135L + NcoI.
  • Leaf disks bombarded with cobG43 V + Ncol were designed for 50 ⁇ M AA after two days of rest, leaf disks bombarded with cobF135L + NcoI for 50 ⁇ M Myx. After one week, the concentration was increased to 100 ⁇ M each. After two weeks at 100 ⁇ M plus 50 ⁇ M SHAM. After three weeks, the promising callus approaches were transferred to 200 ⁇ M AA or Myx plus 100 ⁇ M SHAM. After five weeks, SHAM was also increased to 200 ⁇ M.
  • the selection marker could be further improved by introducing a further mutation into the plasmids used, whereby a Sphl interface present in the native cob gene is switched off, since this enables a PCR reaction to be used to distinguish between native cob and mutated cob.
  • the DNA isolated from the regenerated callus can then be digested completely with SphI.
  • the transgenic cob is retained and can still be amplified using PCR.
  • An exchange of nucleotide T264 for C would open this possibility.
  • FIG. 1 shows the fusion constructs of hexokinase 1 (Hxkl) with the green fluorescent protein (GFP), in which the Hxkl content was of different sizes.
  • HxklN the green fluorescent protein
  • HxklN the N-terminus of Hxkl, comprising the putative signal peptide, was fused to GFP.
  • Hxkl the entire sequence coding for Hxkl was combined with that for GFP.
  • HK1 stands for Hxkl and HK1N for HxklN.
  • Figure 2 shows an electron micrograph of Hxkl :: GFP organelles.
  • aggregated mitochondria can be seen in the middle, peroxisomes in the upper right corner.
  • peroxisomes in the upper right corner.
  • aggregated mitochondria In the right picture are aggregated mitochondria and
  • FIGS. 3 a and b (different magnifications) likewise show electron micrographs of Hxkl :: GFP organelles, it being clearly evident that the formation of the mitochondrial conglomerates appears to be due to a disturbed mitochondrial division.
  • FIG. 4 shows EM images of mitochondrial conglomerates.
  • the left complex has a size of at least 7 ⁇ m, the right one of at least 10 ⁇ m.
  • FIG. 5 shows the construction of the plasmids used for the selection of transgenic mitochondria.

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Abstract

L'invention concerne un procédé pour réaliser des agglomérations de mitochondries, afin de faciliter une transformation subséquente de celles-ci. Ce procédé consiste à réaliser l'expression, dans les cellules contenant lesdites mitochondries, d'un premier composant protéique, apte à l'ancrage dans la membrane extérieure des mitochondries, et d'un deuxième composant protéique, capable de protéger stériquement les déterminants à la surface des mitochondries.
PCT/EP2002/001737 2001-02-19 2002-02-19 Procede pour realiser et transformer des agglomerations de mitochondries WO2002066661A1 (fr)

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DE2001107677 DE10107677A1 (de) 2001-02-19 2001-02-19 Verfahren zur Erzeugung und Transformation von Mitochondrien-Konglomeraten
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Cited By (2)

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GB2465748A (en) * 2008-11-25 2010-06-02 Algentech Ltd Plant mitochondrial transformation method
KR20190124656A (ko) * 2018-04-26 2019-11-05 주식회사 파이안바이오테크놀로지 개질된 미토콘드리아 및 이의 용도

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2465748A (en) * 2008-11-25 2010-06-02 Algentech Ltd Plant mitochondrial transformation method
GB2465748B (en) * 2008-11-25 2012-04-25 Algentech Sas Plant cell transformation method
US9663792B2 (en) 2008-11-25 2017-05-30 Algentech Sas Plant mitochondria transformation method
KR20190124656A (ko) * 2018-04-26 2019-11-05 주식회사 파이안바이오테크놀로지 개질된 미토콘드리아 및 이의 용도
KR102126199B1 (ko) 2018-04-26 2020-06-24 주식회사 파이안바이오테크놀로지 개질된 미토콘드리아 및 이의 용도
KR20200077476A (ko) * 2018-04-26 2020-06-30 주식회사 파이안바이오테크놀로지 개질된 미토콘드리아 및 이의 용도
KR102533834B1 (ko) 2018-04-26 2023-05-19 주식회사 파이안바이오테크놀로지 개질된 미토콘드리아 및 이의 용도

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