DE10107677A1 - Process for the creation and transformation of mitochondrial conglomerates - Google Patents

Abstract

The invention relates to a method for producing clusters of mitochondria in order to simplify the subsequent transformation of said mitochondria. The production of the clusters of mitochondria includes the expression, in the cells containing the mitochondria, of a first protein component which is capable of anchoring in the outer membrane of the mitochondria, and of a second protein component which can sterically protect the determinants on the surface of the mitochondria.

Description

The invention relates to a method for producing mitochondrial conglomerates for facilitated subsequent transformation of the mitochondria, in which the generation of the mitochondrial conglomerates the expression of a first one Protein component leading to anchoring in the outer mitochondrial membrane is capable, and a second protein component, which is able to determine determinants on the Sterically shield the surface of mitochondria into those containing mitochondria Cells includes.  

The expression of the protein components mentioned, the components of a common (Fusion) protein leads to the formation of mitochondrial conglomerates, which thanks to their Size compared to single mitochondria with foreign nucleic acids can be transformed.

Mitochondria, like plastids, are organelles of eukaryotic Cells. Both the mitochondria and the plastids, especially chloroplasts, contain their own DNA. In general, mitochondria are cylindrical, rare spherical, with a diameter of 0.5 to 1 µm. Like their prokaryotic ancestors divide mitochondria and chloroplasts by constriction. It is imagined Ring made of tubulin-related proteins that constricts while consuming GTP. at Chloroplasts, this ring is made of FtsZ. About the division of mitochondria is less known. In the fully known genomes of Caenorhabditis elegans and Saccharomyces cerevisiae, no ftsz homologues were found that were suitable for one mitochondrial division could be responsible, but probably in the unicellular algae Mallomonas splendens and the unicellular red algae Cyanidioschyzon merolae. In yeast and Animals constrict the mitochondria with the participation of dynamin. Dynamins were previously known to be associated with endocytosis, where they become one Pinching off the endocytosis vesicles.

The mitochondrion is surrounded by two highly specialized membranes, the outer one Membrane and the inner membrane, which are crucial for its activity. Each of the two Lipid bilayers contain a unique protein pattern, and enclose together and define two separate mitochondrial compartments: the inner matrix space and the narrow space between the two membrane systems (inter-membrane space).  

There have been publications on successfully transformed plant mitochondria not before. The transformation of mitochondria is especially for the plant Biotechnology and molecular plant breeding of great interest since the Transformation of cell organelles, which is already possible with larger chloroplasts, offers significant advantages over cell nucleus manipulation. So you watch one Organelle transform a higher expression of the transgene, which usually only is inherited maternally, which is another advantage of the Organell transformation, because for example, there is hardly any spread of the transgene through pollen (Bogorad L. (2000) Trends in biotechnology. 18: 257-263; Daniell H. (1999) Nat. Biotechnol. 17: 855-856).

It is therefore an object of the present invention to enlarge mitochondria or Mitochondrial conglomerates, i.e. aggregations or associations of mitochondria, to generate accessible transformation processes. With transformation processes, which are suitable for the transfer of foreign genes to mitochondria for example micromanipulation or bombardment with DNA.

These and other tasks that will become apparent from the description below solved by the method defined in the main claim. Preferred embodiments are specified in the subclaims.

It has now surprisingly been found that the expression of a (fusion) protein which comprises two components, namely a first, which anchors the protein in the outer mitochondrial membrane, and a second one that is able Sterically shielding determinants on the surface of mitochondria, the formation of Mitochondrial conglomerates. With the phenomenon now observed for the first time it is not a coincidental event, but a reproducible one Way to provide larger mitochondrial structures, which due to their size means conventional transformation methods can be genetically manipulated.  

The possibility of larger mitochondrial associations through targeted expression of a Proteins comprising an anchoring component (targeting component) for the outer Mitochondrial membrane and a steric shielding component (shielding Manufacturing component) is described here for the first time.

Without wishing to be bound by a hypothesis, it is currently assumed that the expression of the named protein components an inhibition of the mitochondrial Division causes mitochondrial conglomerates, which are called "Giant mitochondria" could refer to and genetically manipulated due to their size can be.

In a preferred embodiment, the anchoring component is the enzyme hexokinase (HK) or parts of this enzyme that are anchored in the outer mitochondrial membrane are capable. Other preferred anchoring Components are porins or parts thereof, which are used for anchoring in the outer Mitochondrial membrane are capable. Basically, every protein or part is one Protein suitable for a cytoplasmic localization with anchoring in the outer Mitochondrial membrane has.

Hexokinase Hxk 1 from Nicotiana, for example, is an anchoring component tabacum. The sequence of Hxk 1 was published under accession no. AF 118133 published, whereby the description of the sequence in the database shows that Hxk 1 is a chloroplast protein. The sequence of the hexokinase Tobacco was obtained by using a previously characterized hexokinase from spinach (Accession No. AF 118132) a cDNA library was screened from tobacco (Wiese et al. (1999) FEBS Lett. 461: 13-18). The tobacco hexokinase obtained was then obviously not further characterized, but only the description of the spinach  Hexokinase, which is due to a localization of Hexokinase I in the outer Envelope membrane indicates.

In addition to the hexokinase mentioned in the accompanying examples, the person skilled in the art is familiar with further suitable anchoring proteins or parts of proteins that serve as anchoring domain can be used, known. The following are various proteins that are in the outer Mitochondrial membrane are anchored. The expert can use the References below using suitable cloning methods suitable fusion proteins produce and use to generate the desired mitochondrial conglomerates.

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The person skilled in the art can generate suitable gene constructs and transfer them to Plant cells use routine procedures to determine which proteins or parts of proteins are capable of anchoring in the outer mitochondrial membrane, and which proteins because of their amino acid sequence or not because of their sufficient length, especially with parts of proteins, no anchoring in the mediate outer mitochondrial membrane.

The person skilled in the art can also use the proteins described in the prior art, cytoplasmic localization with anchoring in the outer mitochondria Have membrane, especially hexokinases and porins, other suitable proteins  find z. B. by PCR using oligonucleotides known from Nucleic acid sequences are derived, or by hybridization of cDNA or genomic libraries with gene probes, the known nucleic acid sequences correspond.

The second component of the protein, whose expression in plant cells to the desired Formation of mitochondrial conglomerates leads to the so-called shielding components steric shielding of determinants on the mitochondrial surface causes.

In a preferred embodiment, it is green under UV radiation luminous protein Green Fluorescent Protein (GFP), which is often used as a reporter gene or as Selection marker is used. Basically, however, every protein or part is one Suitable protein that is capable of steric shielding of determinants on the Mitochondria surface, especially of receptors.

In the case of this shielding or masking component, too, the person skilled in the art is familiar with Routine experiments can easily determine the suitability of a particular protein or to check part of a protein by expression in plant cells. Important is that the part protruding into the cytosol is able to determine the relevant determinants on the Sterically shield the surface of the mitochondria. It is assumed that the Shielding component should have a size that has a molecular weight of about 20 kDa, preferably 25 kDa and particularly preferably at least 28 kDa equivalent.

The anchoring component and the steric components are preferably in the form a fusion protein expressed in the plant cell, d. H. the fusion protein is made by one chimeric gene construct encoding an artificial assembly of the for the Anchoring component and DNA sequences encoding the steric component  represents. In principle, however, it is not excluded that the expression of a natural occurring protein for the inventive method for producing Mitochondrial conglomerates is suitable. In addition, it is assumed that the Expression of two interacting proteins, one of which as Shielding component acts and the other protein does the job Anchoring component also takes over the desired conglomeration of the Mitochondria causes.

The preparation of suitable gene constructs for fusion proteins, comprising the Anchoring component and the steric component, encode, is the expert common and possible using standard molecular biological methods, see e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

Also the various methods for transferring gene constructs and plasmids Plant cells are well known to those skilled in the art. The same applies to regulatory Sequences that are suitable for the expression of genes in plants, such as. B. constitutive Promoters, tissue and development-specific promoters, inducible promoters, Enhancer sequences and other regulatory sequences that transcribe and Translation of a coding DNA sequence operatively linked to a promoter region control in transformed plant cells.

In a preferred embodiment, the expression of the Fusion protein controlling promoter to a constitutive promoter, such as for example the 35S RNA promoter from CaMV, or a leaf-specific one Promoter.  

Prepare for the introduction of foreign genes into higher plants or their cells a large number of cloning vectors are available which provide a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. examples for such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence, in the present case the fusion of (i) sequences which are necessary for the Code anchoring component, with (ii) sequences for the steric Coding shielding component can at a suitable restriction interface in the Vector are introduced. The plasmid obtained is then used for the transformation of E. coli cells used. Transformed E. coli cells are in a suitable medium grown and then harvested and lysed, and the plasmid is recovered. As Analysis method for the characterization of the plasmid DNA obtained are in general restriction analyzes, gel electrophoresis and other biochemical molecular biological methods used. After each manipulation, the plasmid DNA cleaved and DNA fragments obtained are linked to other DNA sequences.

There are a large number of for the introduction of DNA into a plant host cell Techniques are available, the person skilled in the art the most suitable method without Can identify difficulties. These techniques include plant transformation T-DNA cells using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation medium, the fusion of protoplasts, the injection, the Electroporation, the direct gene transfer of isolated DNA into protoplasts, the introduction of DNA using the biolistic method and other options that have been around since are well established for several years and are part of the usual repertoire of professionals in the plant molecular biology or plant biotechnology belong.

In the injection and electroporation of DNA into plant cells, none per se made special demands on the plasmids used. The same applies to the direct gene transfer. Simple plasmids such as e.g. B. pUC derivatives used  become. However, whole plants are to be regenerated from cells transformed in this way the presence of a selectable marker gene is recommended. Those skilled in the art are known selection marker, and it is not a problem for him, a suitable one Select marker.

Depending on the method of introducing desired genes into the plant cell, additional DNA Sequences may be required. Are z. B. for the transformation of the plant cell the Ti or Ri plasmid is used, at least the right boundary, but often the right and left delimitation of the T-DNA contained in the Ti and Ri plasmid as Flank area with the genes to be introduced. Will for the Transformation Agrobacteria used, the DNA to be introduced into special plasmids be cloned, either in an intermediate or in a binary vector. The intermediate vectors may be due to sequences homologous to sequences in the T-DNA are, by homologous recombination in the Ti or Ri plasmid of the agrobacteria to get integrated. This also contains the vir- necessary for the transfer of the T-DNA Region. Intermediate vectors cannot replicate in agrobacteria. By means of a Helper plasmids can transfer the intermediate vector to Agrobacterium tumefaciens become (conjugation). Binary vectors can be found in E. coli as well as in Agrobacteria replicate. They contain a selection marker gene and a linker or polylinker, which be framed by the right and left T-DNA border regions. You can go straight to the Agrobacteria are transformed. The agrobacterium serving as the host cell is said to be Plasmid carrying a vir region included. The vir region is for the transfer of T-DNA necessary in the plant cell. Additional T-DNA may be present. That like that transformed agrobacterium is used to transform plant cells. The Use of T-DNA for the transformation of plant cells has been intensively investigated and sufficient in well-known overview articles and manuals for Plant transformation has been described. For the transfer of DNA into the plant cell can plant explants expediently with Agrobacterium tumefaciens or  Agrobacterium rhizogenes can be cultivated. From the infected plant material (e.g. Leaf pieces, stem segments, roots, but also protoplasts or suspension cultivated Plant cells) can then be in a suitable medium, which is antibiotics or biocides for the selection of transformed cells, can regenerate whole plants again become.

Once the introduced DNA is integrated in the genome of the plant cell, it is there in the Usually stable and remains in the progeny of the originally transformed cell receive. It usually contains a selection marker, which is the transformed one Plant cells resistance to a biocide or an antibiotic like kanamycin, G 418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, Gentamycin or Phosphinotricin u. a. taught. The individually chosen marker should hence the selection of transformed cells over cells to which the introduced DNA missing, allow. Alternative markers are also suitable for this, such as nutritive markers, Screening markers. Of course, selection markers can also be dispensed with entirely which, however, goes hand in hand with a fairly high need for screening. If Strategies are also available to those skilled in the art if marker-free transgenic plants are desired Available, which allow a subsequent removal of the marker gene, e.g. B. Cotransformation, sequence-specific recombinases.

The regeneration of the transgenic plants from transgenic plant cells takes place after usual regeneration methods using known nutrient media. The so Plants obtained can then be subjected to conventional procedures including molecular biological methods, such as PCR, blot analysis, for the presence of the Introduced nucleic acid for the fusion protein consisting of Anchoring component and shielding component, coded, are examined.  

The transgenic plant or the transgenic plant cells can be any act any monocot or dicot plant or its plant cell. Preferably are useful plants or cells of useful plants. Acts particularly preferred it is corn, rice, wheat, barley, oats, rye, soybeans, rapeseed, potatoes, tomatoes, tobacco, Sugar beet, pea, banana, pineapple, paprika, yams, cassava, cotton.

Those transformed with the fusion gene construct coding for the fusion protein Plant cells show the phenomenon according to the invention of the formation of mitochondria Conglomerates caused by an inhibition of the naturally occurring Mitochondrial division. This inhibition is achieved by anchoring the fusion protein in the outer mitochondrial membrane, caused by the anchoring component of the Fusion protein, and shielding of determinants on the mitochondrial surface, caused by the steric component of the fusion protein, mediated.

The mitochondrial conglomerates of the transformed plant cells can because of their Size can be genetically modified using known transformation methods.

The "particle bombardment" or the is particularly suitable as a transformation method Microinjection, but also other methods are conceivable if they are for example the transformation of plastids or protoplasts are suitable or proven for these objects to have.

The particle bombardment method was introduced in 1987 (Klein et al., Nature 327: 70-73) published and has meanwhile entered the relevant as the standard method Technical books found. The method has now also been applied to mitochondria of Saccharomyces cerevisiae and Chlamydomonas reinhardtii (see e.g. Johnston et al. (1988) Science 240: 1538-1541; Boynton et al. (1996) Methods Enzymol. 264: 279-296; Butow and Fox (1990) Trends Biochem, Bei. 15: 465-468).  

Animal mitochondria have already been successfully transformed by electroporation (Collombet et al. (1997) J. Biol. Chem. 272: 5342-5347).

Known for years as a means of nuclear transformation (Crossway et al. (1986) Mol. Gene. Genet. 202: 79-85), the method of injecting DNA using a fine Capillaries have now been refined to such an extent that chloroplasts can also be transformed. (Knoblauch et al. (1999) Nat. Biotechnol. 17: 906-909.) The inventive Mitochondrial conglomerates partially exceed the size of chloroplasts, so that too whose transformation should be carried out by means of microinjection.

After successful transformation of the mitochondria, they must be analogous to transgenic Chloroplasts can be selected by backcrossing. A backcross of those Plants with transformed mitochondria is necessary to determine the phenotype of the Eliminate mitochondrial conglomerates. The back crossing can be facilitated by the gene leading to the conglomerates with a negative selection marker is cotransformed. This negative selection marker kills under appropriate conditions all plants transformed in the core. Along with conditions that are positive Enable selection of transformed mitochondria, only plants can survive which have transformed mitochondria and their gene leading to conglomerates together negative selection marker was crossed out.

Examples of negative selection markers are e.g. B. codA (cytosine deaminase; Stougaard (1993) Plant J. 3: 755-761) and dhlA (haloalkane dehalogenase; Naested et al. (1999) Plant J. 18: 571-576).

The invention thus also relates to plant cells and plants which have mitochondria, which were transformed by means of the method according to the invention, and propagation material  and harvest products of these plant cells or plants, for example fruits, Seeds, tubers, rhizomes, seedlings, cuttings, etc.

The present invention is illustrated in the following examples, which are only the Serve to illustrate the invention and are not to be understood in any way as a limitation are explained.

Examples General cloning procedures

Cloning procedures such as B. restriction cleavages, DNA isolation, agarose Gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to Nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, culture of bacteria, sequence analysis of recombinant DNA according to Sambrook et al. (1989, vide supra).

The transformation of Agrobacterium tumefaciens was carried out according to the method of Höfgen and Willmitzer (Nucl. Acids Res. (1988) 16, 9877). The cultivation of the Agrobacteria occurred in YEB medium (Vervliet et al. (1975) J. Gen. Virol. 26, 33).

Bacterial strains and plasmids

E. coli (XL-1 Blue) bacteria were from Stratagene (La Jolla, California, USA) based. The Agrobacterium strain (C58C1 with the plasmid pGV 3850kan) was developed by Debleare et al. (1985, Nucl. Acids Res. 13, 4777)  described. For the cloning, the vectors pCR-Blunt (Invitrogen, Carlsbad, California, USA), pBluescript SK- (Stratagene) and pBinAR (Höfgen and Willmitze (1990) Plant Sci. 66: 221-230) is used.

tobacco transformation

For the tobacco transformation of tobacco plants (Nicotiana tabacum L.cv. Samsun NN) 10 ml of an overnight culture of Agrobacterium tumefaciens grown under selection centrifuged, the supernatant discarded, and the bacteria in the same volume Resuspended medium free of antibiotics. Leaf disks were placed in a sterile petri dish sterile plants (diameter approx. 1 cm) bathed in this bacterial solution. Subsequently the leaf disks were placed in petri dishes on MS medium (Murashige and Skoog (1962) Physiol. Plant 15, 473) with 2% sucrose and 0.85 Bacto agar. After 2 days The leaf disks were incubated in the dark at 25 ° C. on MS medium at 100 mg / l Kanamycin, 500 mg / l Claforan, 1 mg / l benzylaminopurine (BAP), 0.2 mg / l naphthyl acetic acid (NAA), 1.6% glucose and 0.85 Bacto agar transferred and the cultivation (16 h Light / 8 h dark) continued. Growing sprouts were on hormone-free MS Medium transferred with 2% sucrose, 215 mg / l Claforan and 0.8% Bacto agar.

Cloning of the chimeric transgenes

Two fusion constructs of hexokinase 1 (Hxk1, see above) were generated with the Green Fluorescent Protein (GFP), in which the Hxk1 content was different. The fusion constructs of Hxk1 and GFP are shown in Fig. 1. In the shorter construct HxK1N :: GFP (N stands for N-terminus), only the N-terminus of Hxk1, comprising the putative signal peptide, was fused with GFP. In the larger construct HxK1 :: GFP, the entire sequence coding for Hxk1 was combined with that for GFP.

The fusion partners GFP and Hxk1 and Hxk1N were created using the polymerase chains reaction (polymerase chain reaction, PCR) cloned. The polymerization steps were in an automated T3 thermal cycler (Biometra, Göttingen, Germany) according to the below specified programs. The PCR constructs were in the vector pCR Blunt (Invitrogen) increased. The Hxk1 subfragments were then labeled as BamHI / EcoRI Restriction fragments ligated into the vector pBluescript SK- (Stragagene). The GFP fragment was connected as an EcoRI / SalI restriction fragment. The whole construct was then cut out as a BamHI / SalI fragment and ligated into the binary vector pBinAR.

The following oligonucleotides and templates were used for the PCR amplifications.

(The restriction sites for the enzymes EcoRI, BamHI and SalI are highlighted).

A HK1 cDNA clone was used as a template for Hxk1, which clone from a cDNA library was isolated, which is identical to that from which the by Wiese et al. published light clone Hxk1 was isolated (Wiese et al. (1999) FEBS Lett. 461: 13-8.).

The vector pBin-mGFPS-ER was used as the template for GFP (Siemering K.R. et al. (1996), Curr. Biol. 6: 1653-1663).  

The composition of the PCR reaction batches and the PCR used in each case Program are given below.

For Hxk1N

program

For Hxk1

program

For GFP

program

The nucleic acid sequences of the fusion constructs described above are together with the amino acid sequences of the fusion proteins encoded by the fusion constructs below specified.

Nucleic acid sequence of the fusion construct HxK1N :: GFP

The first 25 bases correspond to the sequence of the PCR primer HK9GFP5, including the BamHI restriction site (italic). The ATG start codon from Hxk1 is highlighted. The EcoRI restriction site via which the GFP fragment connects to the Hxk1 fragment connected, is also shown in italics. The last 29 bases result from the PCR primer K76 used (counter strand, including SalI- Restriction site).  

Amino acid sequence of the fusion protein Hxk1N :: GFP

The EcoRI fusion results in amino acids 43-44, EF. The EcoRI interface GAATTC is already in the GFP template pBin-mGFP5-ER, which means that Endoplasmic reticular signal sequence (pBin-mGFP5-ER) was connected upstream. This However, signal sequence was not used in the context of the invention and the EcoRI Nucleotides are not necessary for GFP to function (see Access Numbers U87974 with, and U87973 without this restriction site). The sequence of Hxk1 on that Digit is GAATTT, which is also translated to EF. The amino acids EF can therefore still count towards the hexokinase sequence.

Nucleic acid sequence of the HxK1 :: GFP fusion construct

In HxK1 :: GFP, the Hxk stop codon TAG was mutated by EcoRI in GAA. The Amino acids EF are at positions 498-499.

Amino acid sequence of the fusion protein HxK1 :: GFP

Confocal laser scanning microscopy

The epidermis was peeled off from the underside of a leaf and the CLSM 410 Zeiss (Jena, Germany) irradiated with light of the wavelengths 488 and 543 nm. The Emissions were limited to 510 to 525 nm by a bandpass filter. GFP protein became visible in light green.

Transfer and expression of the fusion proteins HxK1 :: GFP and Hxk1N :: GFP in tobacco

The fusion constructs were transferred to tobacco cells and transgenic as indicated above Plants regenerate. Epidermis samples of the transgenic plants showed in fluorescence Microscope glowing green signals of the GFP. What was surprising at first was that the both constructs showed different distribution patterns of GFP. While the little one Construct several small glowing GFP signals caused by the larger one Construct significantly larger luminous areas, comparable in size to the cell nucleus, marked. The electron microscopic examination of the plant tissue then showed that the large GFP-marked areas were collections of mitochondria that were found in were aggregated to varying degrees. Associated with these aggregates were also Peroxisomes. In the plants transformed with the smaller construct, Hxk1N :: GFP the mitochondria also showed an affinity for each other, but in a weaker way Extent than Hxk1 :: GFP.  

The localization of GFP on the outer mitochondrial membrane led to Mitochondrial aggregation. The degree of aggregation was of the size of the GFP fusion protein dependent. The chimeric protein Hxk1 :: GFP led to large ones Conglomerates of mitochondria and peroxisomes, while the smaller fusion protein HxK1N :: GFP only generated an affinity for both organelle types. While the effect of the larger construct was already detectable in the light microscope, was the small effect of HxK1N :: GFP can only be seen under the electron microscope.

It is assumed that the formation of the mitochondrial conglomerates is due to the fact that the large amount of foreign proteins anchored in the outer mitochondrial membrane disturbs the complete division of the organelles, and the presence of the fusion protein thus limits the division of the mitochondria by constriction - or prevented. The electron micrographs shown in Fig. 3 also speak for this.

Transformation of the mitochondrial conglomerates

In the first published transformation of yeast mitochondria, a mutation in the mitochondrial gene oxi3, coding for the largest subunit of cytochrome oxidase (COXI), was corrected by homologous recombination with the WT gene. Because the cells regained the ability to breathe, they could be screened. The transformation of the mitochondrial conglomerates according to the invention described here is based on oligomycin resistance mediated by mitochondrial transformation. Oligomycin specifically inhibits the mitochondrial F _O F _l ATPase (hence the name FO for oligomycin-sensitive; this designation was then retained for the chloroplastid ATPase, although it is not sensitive). In the mitochondrial genome oligomycin-resistant yeast and hamster cells, 6 mutations were found in the gene for the subunit, which are probably responsible for the resistance. (Breen et al. (1986) J. Biol. Chem. 261: 11680-11685; Holmans et al. (1987) Somat. Cell. Mol. Genet. 13: 347-353; John and Nagley (1986) FEBS Lett. 207: 79-83). A mutation in the mitochondrial gene for subunit 9 was made responsible for a further form of resistance to oligomycin, or venturicidin and ossamycin in yeast. (Galanis M et al. (1989) FEBS Lett. 249: 333-336; Nagley et al. (1986) FEBS Lett. 195: 159-163). Both genes are also widespread in plant mitochondria, so that a transformation protocol can be built up on resistance imparted thereby.

The first step to a reproducible protocol is a mutated form of a of the two named genes that confer resistance to oligomycin into a mitochondrion contribute. At it by particle bombardment or by microinjection. Both Methods are used analogously to plastid transformation. The screening on Resistance to oligomycin provides proof of a successful transformation.

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 e.g. 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. There are two strategies:

• a) The original gene for ATPase subunit is replaced by the marker gene. By the transgene also integrating here may become important Genes replaced.  
• b) the chimeric gene integrates at an unimportant location in the mitochondrial genome, which ensures the survival of the original genes. The marker gene for the Oligomycin resistance does not replace the original gene for ATPase Subunit, but this also remains. So that would have transformed mitochondrial genome two genes for the ATPase subunit, only one Confers resistance to oligomycin. The disadvantage here would possibly be a diminished one Resistance.

Thorsness and Fox managed to insert a foreign gene (ura3) into the mitochondrial genome of yeast to integrate. This, however, without a promoter, since it was only examined whether this gene from mitochondrial into the nuclear genome. (Thorsness and Fox (1990) Nature 346: 376-379). The same is true according to the protocol described above and also above mentioned transformation methods the integration of foreign DNA also in plant Mitochondria possible.

characters

Fig. 1 shows the fusion constructs of hexokinase 1 (Hxk1) with the Green Fluorescent Protein (GFP) in which the Hxk1 proportion was different in size. In the shorter construct Hxk1N :: GFP, only the N-terminus of Hxk 1, comprising the putative signal peptide, was fused to GFP. In the larger construct Hxk1 :: GFP, the entire sequence coding for Hxk1 was combined with that for GFP. In Fig. 1, HK1 stands for Hxk1 and HK1N for Hxk1N.

Fig. 2 shows an electron micrograph of Hxk1 :: GFP-organelles. In the left picture, aggregated mitochondria can be seen in the middle, peroxisomes in the upper right corner. The right picture shows aggregated mitochondria and chloroplasts. The black dots are gold-labeled antibodies against GFP.

Figures 3a and b (different magnifications). Also show electron micrographs of HxK1 :: GFP organelles, it being clearly seen that the formation of the mitochondria conglomerates to impaired mitochondrial division appears to be due.

Claims (10)

1. A method for producing mitochondrial conglomerates comprising the Expression of a first protein component leading to anchoring in the outer Mitochondrial membrane is capable of, and a second protein component leading to a steric shielding of determinants on the surface of mitochondria in the Is able to plant cells. 2. The method of claim 1, wherein the first and second protein components are in the form of a fusion protein can be expressed in the plant cell. 3. The method according to claim 1 or 2, wherein it is for anchoring in the outer mitochondrial membrane enabled protein component around the enzyme hexokinase or is a porin or parts of this enzyme or a porin, for anchoring in the outer mitochondrial membrane. 4. The method according to any one of the preceding claims, wherein it is for steric shielding of determinants on the surface of mitochondria qualified protein component are the green fluorescent protein or parts thereof, for the steric shielding of determinants on the surface of mitochondria in are able. 5. The method according to any one of the preceding claims, wherein the steric Shielding of determinants on the surface of mitochondria Protein component has a size that has a molecular weight of at least 20 kDa, preferably of at least 25 kDa and particularly preferably of at least 28 kDa equivalent.   6. A process for the production of transgenic plants, comprising the production of Mitochondrial conglomerates by the method according to one of claims 1 to 5. 7. A method for producing transgenic plants by means of mitochondrial transformation, comprising the steps

• a) generation of mitochondrial conglomerates by a method according to one of the preceding claims,
• b) transformation of the mitochondria with a nucleic acid molecule
• c) Crossing out the phenotype of the conglomerated mitochondria.

8. The method according to claim 7, wherein the transformation in step b) by means of biolistic methods, in particular particle bombardment, by means of microinjection or by electroporation. 9. Use of mitochondrial conglomerates, produced by a process according to one of claims 1 to 5 for the transformation of mitochondria. 10. Transgenic plant produced by a method according to one of claims 6 till 8.