EP2007894A2 - Method for modifying the atp/adp ratio in cells - Google Patents

Abstract

The invention relates to a method for modifying the ATP/ADP ratio in a cell, tissue, organ, microorganism or plant by modifying the hemoprotein activity in the cell. The invention also relates to the use of said method.

Description

 A method for altering the ATP-ADP ratio in cells

The invention relates to a method for altering the ATP / ADP ratio in a cell, tissue, organ, microorganism or plant by altering the hemoprotein activity in the cell and its use.

Adenosine triphosphate (ATP) is formed in the process of both photosynthesis and respiration from ADP (adenosine diphosphate) and high-energy phosphate bonds. These are endergonic reactions. The energetic ATP is hydrolyzed by ATPases releasing energy. Since most processes in the cell are endergonic, they become possible only through the coupling with a second exergonic reaction, which in most cases involves the hydrolysis of ATP.

The hydrolysis of ATP to ADP serves as a driving force in many biochemical processes such. B. active transport through membranes, biosynthesis of lipids, proteins, carbohydrates or nucleic acids.

Thus, ATP in the cell is an energy carrier that provides the energy for ultimately any activity of the cell or organism. Each organism thus uses ATP as its primary source of energy. As a result, ATP plays a key role in the metabolism of the cell. On the other hand, ATP has a very short half life ("lifetime") and therefore virtually no storage capacity.

The ATP-ADP ratio is an important parameter of energy metabolism. A high ATP-ADP ratio means an energy surplus. When there is a lack of energy in the cell, the intracellular ATP reserves are consumed and the ATP-ADP ratio is shifted towards ADP.

The expression of hemoglobin or related proteins is known in the art.

US Patent 6,372,961 discloses the expression of genes coding for hemoglobin, thereby increasing oxygen metabolism in plants. This increased oxygen or ATP content may affect biosynthesis in the plants.

From WO 98/12913 a method for increasing oxygen assimilation is known which is based on expression of hemoglobin proteins. Further, this document discloses that an increase in the production of secondary metabolites can be attributed to a simultaneous increase in the ATP concentration. Furthermore, it is known from WO 00/00597 that the expression of non-symbiotic hemoglobin in cells leads to an increase in the ATP content. The expression of hemoglobin and related proteins was used according to WO 99/02687 A to increase the iron content in cells.

In WO 2004/057946 A, a higher yield of starch and oil in plants is achieved by the expression of leghemoglobin.

From the document WO 2004/087755 a method is known for increasing the stress resistance of plants and the yield obtained therefrom based on the expression of class two vegetable.

The expression of leghemoglobin in plant cells is also known from Barata et al .: (Plant Science, Vol.155, June 2000, 193-202), wherein oxygen availability is examined.

An increase in the ATP-ADP ratio is not known from the prior art.

Object of the present invention is to provide a method by which the cell or the organism more ATP and thus more energy is available. In particular, ATP should also be used as energy storage, that is, it should be achieved an increase in the ATP-ADP ratio. Another object of the present invention is the energy thus provided specifically for the synthesis of fatty acids, in particular alpha-linolenic acid (ice, ice, cis-9,12,15-Octadecatriensäuere)

These objects are achieved by changing the activity of at least one hemoprotein in the method according to the invention for altering the ATP-ADP ratio in a cell, tissue, organ, microorganism or plant.

Surprisingly, it has been found that the modification of the activities of at least one hemprotein produces cells, organs, tissues, microorganisms or plants which have an increased ATP-ADP ratio.

By the ATP-ADP ratio is meant the ratio of the concentration of ATP to the concentration of ADP. The concentrations can be determined by the customary methods known to the person skilled in the art, for example by 31 P-NMR spectroscopy in intracellular measurement or as described below in the examples. For the purposes of the present invention, the term cell comprises cells, parts of plants such as organs or tissues, as well as whole plants and microorganisms.

Hemoproteins are proteins capable of binding oxygen via a prosthetic group, such as non-symbiotic hemoglobin, myoglobin or leghemoglobin, preferably leghemoglobin and nonsymbiontic hemoglobin, most preferably leghemoglobin.

"Hemoprotein activity" means the ability of the polypeptide to bind oxygen to the prosthetic group (hem). These are understood according to the invention iron-II complexes of protoporphyrin. Altering the activities of a hemprotein in a cell means the ability to bind more or less oxygen in the cell compared to cells of the wild type of the same genus and species to which the methods of the invention have not been applied, all other conditions being equal (such as culture conditions, age the cells etc.). The change, increase or decrease, preferably increase, in comparison to the wild type is at least 1%, 2%, 5%, 10%, preferably at least 10% or at least 20%, particularly preferably at least 40% or 60%, completely more preferably at least 70% or 80%, most preferably at least 90%, 95% or more. In one embodiment of the present invention, the ATP-ADP ratio is at least 200%, preferably 300%, more preferably at least 400% or more, based on the wild-type ATP-ADP ratio.

The comparison is preferably carried out under analogous conditions. "Analogous conditions" means that all framework conditions such as culture or breeding conditions, assay conditions (such as buffer, temperature, substrates, concentration, etc.) are kept identical between the experiments to be compared and the approaches are determined solely by the activity of Distinguish Hemproteins.

Altering according to the invention means a de novo introduction of the activity of a polypeptide according to the invention into a cell, tissue, organ, microorganism or plant or a reduction or, preferably, an increase in an already existing activity of the polypeptide according to the invention. In one embodiment of the present invention, the concentration of the hemoproteins is increased.

Altering the activity of a hemoprotein can be achieved by altering the structure of the proteins, by altering the stability of the hemoproteins or by altering the concentration of hemoproteins in a cell. A preferred variant of the present invention is characterized in that the activities of a hemoprotein, preferably a non-symbiotic hemoglobin or a leghemoglobin is increased.

More preferably, the activity of a polypeptide encoded by a nucleic acid molecule comprising at least one nucleic acid molecule selected from the group consisting of: a) nucleic acid molecule encoding a polypeptide comprising in SEQ ID NO 2, 4, 6, 8, 10 , 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; b) nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34 , 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) Nucleic acid molecule obtained by mixing a nucleic acid molecule from a cDNA database or from a genome database using the primer according to

Sequence Nos. 41 and 42 amplifies f) a nucleic acid molecule encoding a polypeptide having hemoprotein activity which hybridizes under stringent conditions to a nucleic acid molecule according to (a) to (c); and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45.

In a preferred embodiment, the activities of the hemoprotein according to the invention are increased by expression, preferably overexpression in comparison to the wild type as described above, of at least one nucleic acid molecule comprising at least one nucleic acid molecule selected from the group consisting of: a) nucleic acid molecule which encodes a polypeptide comprising those shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38 , 39 and / or 40 shown sequence; b) nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34 , 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) Nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45 includes.

"Nucleic acids" means biopolymers of nucleotides which are linked together via phosphodiester bonds (polynucleotides, polynucleic acids) Depending on the type of sugar in the nucleotides (ribose or deoxyribose), a distinction is made between the two classes of ribonucleic acids (RNA) and deoxyribonucleic acids ( DNA).

The terms "protein" and polypeptide "are synonymous and mutually interchangeable in the sense of the present invention. In a preferred embodiment of the present invention, transformed cells are produced by expression of a non-symbiotic hemoglobin, preferably plants having an increased ATP-ADP ratio.

Non-symbiotic hemoglobin belongs to the family of hemoglobin proteins whose function is reversible oxygen binding and supply. In contrast to leghemoglobin, it does not occur in the root nodules of legumes (legumes). You are u.a. involved in the detoxification of nitrite oxide and in the detection of oxygen availability.

In a further preferred embodiment of the present invention, cells transformed by expression of a leghemoglobin are produced, preferably plants having an increased ATP-ADP ratio.

Leghemoglobin belongs to the family of hemoglobin proteins whose function is reversible oxygen binding and supply. It comes from root nodules of legumes (legumes) and is an isolable red substance that resembles the vertebrate myoglobin. Due to the reversible binding of O2, leghemoglobin can ensure the high oxygen demand during nitrogen fixation by the nodule bacteria. The apoprotein is formed by the plant cells and the hem by the bacteria (Source: CD Römpp Chemie Lexikon - Version 1.0, Stuttgart / New York: Georg Thieme Verlag 1995). Expression in the present application means the transfer of genetic information starting from DNA or RNA into a gene product (polypeptide or protein, here leghemoglobin) and is also intended to include the term overexpression, by which is meant enhanced expression, such that the foreign protein or the naturally occurring protein is produced to a greater extent or makes up a large part of the total protein content of the host cell.

The expression of the hemoproteins according to the invention is achieved by transformation of cells.

"Transformation" describes a process for introducing heterologous DNA into a pro- or eukaryotic cell. With a transformed cell, not only the product is the transformation process per se, but also all the transgenic ones

Progeny of the transgenic organism produced by the transformation. Transformation thus means the transfer of genetic information into an organism, in particular a plant. Including all known to those skilled opportunities for the introduction of information, z. As microinjection, electroporation, particle bombardment (particle bombardment), Agrobakterien or Che- mediated uptake (eg, polyethylene glycol-mediated DNA uptake or via the silicon carbonate fiber technique). The genetic information can be z. B. as DNA, RNA, plasmid and otherwise introduced into the cells and incorporated either by recombination into the host genome, in free form or independently present as a plasmid.

The transformation can be carried out by means of vectors containing the abovementioned nucleic acid molecules, preferably vectors containing expression cassettes which contain the abovementioned nucleic acid molecules. An expression cassette contains a nucleic acid sequence according to the invention operably linked to at least one genetic control element, such as a promoter, and advantageously to another control element, such as a terminator. The nucleic acid sequence of the expression cassette may be, for example, a genomic or a complementary DNA sequence or an RNA sequence and semisynthetic or fully synthetic analogs thereof. These sequences may be linear or circular, extra-chromosomal or integrated into the genome. The corresponding nucleic acid sequences can be prepared synthetically or obtained naturally, or contain a mixture of synthetic and natural DNA components, as well as consist of different heterologous gene segments of different organisms. The term "genetic control sequences" is to be understood broadly and means all those sequences which have an influence on the production or the function of the expression cassette according to the invention. Genetic control sequences, for example, modify transcription and translation in prokaryotic or eukaryotic organisms. Preferably, the expression cassettes according to the invention 5'-upstream of the respective transgene to be expressed nucleic acid sequence comprise a promoter with one of the specificity described above and 3'-downstream terminator sequence as an additional genetic control sequence, and optionally further conventional regulatory elements, respectively functionally linked to the transgenic nucleic acid sequence to be expressed.

In one embodiment of the present invention, homologs of the nucleic acid molecules of the invention are used.

"Homology" between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence / polypeptide sequence over the entire sequence length as determined by comparison with the BESTFIT alignment (according to Needleman and Wunsch 1970, J. Mol. Biol. 48; 443-453 ) setting the following parameters for amino acids Gap Weight: 50 Length Weight: 3

Average Match: 10,000 Average Mismatch: -9,000

is calculated,

and the following parameters for nucleic acids

Gap Weight: 50 Length Weight: 3

Average Match: 10,000 Average Mismatch: 0,000

Instead of the term "homologous" or "homology", the term "identity" is used synonymously in the following.

In one embodiment of the present invention, functional equivalents of SEQ ID NO: 1, 3, 5 are used. Inventive functional equivalents of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31 derived by backtranslating an amino acid sequence having an identity of at least 40%, 50%, 60%, 61%, 62%, 63%, 64%, 65% or 66%, preferably at least 67%, 68%, 69%, 70% , 71%, 72% or 73%, preferably at least 74%, 75%, 76%, 77%, 78%, 79% or 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 89%, 90%, 91%, 92% or 93%, more preferably at least 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40. Functional equivalents of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31 are described by encodes an amino acid sequence having an identity with SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 of at least 40%, 50%, 60%, 61%, 62%, 63%, 64%, 65% or 66% preferably at least 67%, 68%, 69%, 70%, 71%, 72% or 73%, preferably at least 74%, 75%, 76%, 77%, 78%, 79% or 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92% or 93% more preferably at least 94%, 95%, 96%, 97%, 98% or 99%.

"Functional equivalents" here describe nucleic acid sequences which hybridize under standard conditions with a nucleic acid sequence or parts of a nucleic acid sequence and are capable of effecting the expression of the hemoproteins in a cell or an organism.

For hybridization, it is advantageous to use short oligonucleotides with a length of about 10-50 bp, preferably 15-40 bp, for example of the conserved or other regions which are known to the person skilled in the art via comparisons with other related genes. ter way can be determined used. However, it is also possible to use longer fragments of the nucleic acids according to the invention with a length of 100-500 bp or the complete sequences for the hybridization. Depending on the nucleic acid / oligonucleotide used, the length of the fragment or the complete sequence or whichever type of nucleic acid, ie DNA or RNA, are used for the hybridization, these standard conditions vary. For example, the melting temperatures for DNA: DNA hybrids are about 10 ° C lower than those of DNA: RNA hybrids of the same length.

Under standard hybridization conditions, for example, depending on the nucleic acid, temperatures between 42 and 58 ^° C. in an aqueous buffer solution with a concentration between 0.1 to 5 × SSC (1 × SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide such as 42 ° C in 5 x SSC, 50% formamide to understand. Advantageously, the hybridization conditions for DNA: DNA hybrids are advantageously 0.1 x SSC and temperatures between approximately 2O ⁰ C to 65 ⁰ C, preferably between about 3O ⁰ C to 45 ⁰ C. For DNA: RNA hybrids the hybridization conditions are advantageous at 0.1 × SSC and temperatures between about 3O ⁰ C to 65 ⁰ C, preferably between about 45 ⁰ C to 55 ⁰ C. These temperatures for the hybridization are exemplary calculated melting temperature values for a nucleic acid having a length of about 100 Nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant textbooks of genetics, such as Sambrook et al., "Molecular Cloning", CoId Spring Harbor Laboratory, 1989, and can be determined by formulas known to those skilled in the art, for example, depending on the length of the nucleic acids that calculate type of hybrid or G + C content. Further information on hybridization can be found in the following textbooks by the person skilled in the art: Ausubel et al. (eds), 1985, "Current Protocols in Molecular Biology," John Wiley & Sons, New York; Harnes and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

A functional equivalent is furthermore also understood as meaning nucleic acid sequences which are homologous or identical to a defined nucleic acid sequence ("original nucleic acid sequence") to a defined percentage and have the same activity of the original nucleic acid sequences, and in particular also natural or artificial ones Mutations of these nucleic acid sequences. Corresponding definitions can be found at appropriate places in the description. "Mutations" of nucleic acid or amino acid sequences include substitutions (substitutions), additions (additions), deletions (deletion), inversions (alterations) or insertions (insertions) of one or more nucleotide residues, whereby the corresponding amino acid sequence of the target protein is also expressed by subunits. institution, insertion or deletion of one or more amino acids, but overall the functional properties of the target protein are substantially retained.

Furthermore, such nucleotide sequences are also included under the term of the functional equivalent by the present invention, which can be obtained by modification of the nucleic acid sequences SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31 receives. By way of example, such modifications may be made by techniques known to those skilled in the art, such as Site Directed Mutagenesis, Error Prone PCR, DNA Shuffling (Nature 370, 1994, pp. 389-391) or Staggered Extension Process (Nature Biotechnol. 16, 1998, pp. 258-261). The aim of such a modification may e.g. the insertion of additional restriction enzyme sites, the removal of DNA to shorten the sequence, the replacement of nucleotides for codon optimization, or the addition of additional sequences. Proteins which are encoded via modified nucleic acid sequences must still have the desired functions despite deviating nucleic acid sequence.

Functional equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. obtained by chemical synthesis, adapted to the codon use nucleic acid sequences or the amino acid sequences derived therefrom.

Nucleotide sequence is understood to mean all nucleotide sequences which (i) correspond exactly to the sequences shown; or (ii) at least one nucleotide sequence corresponding within the range of the degeneracy of the genetic code to the sequences shown; or (iii) at least one nucleotide sequence which hybridizes to a nucleotide sequence complementary to the nucleotide sequence (i) or (ii) and optionally (iiii) comprises functionally neutral sense mutations in (i). The term "functionally neutral sense mutations" means the replacement of chemically similar amino acids, such. Glycine by alanine or serine by threonine.

According to the invention, modified forms are proteins in which there are changes in the sequence, for example at the N- and / or C-terminus of the polypeptide or in the region of conserved amino acids, but without the function of the protein. These changes can be made in the form of amino acid substitutions by known methods.

According to the invention, the coding regions (structural genes) preceding (5 ^" upstream or upstream) and / or subsequent (3 ^" or downstream) sequences are included. In particular, this includes sequence regions with regulatory function. They can influence transcription, RNA stability or RNA processing as well as translation. Examples of regulatory sequences include promoters, enhancers, operators, terminators or translation enhancers.

A further subject of the present invention is a nucleic acid molecule which codes for a polypeptide which comprises a polypeptide which is encoded by a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of a) nucleic acid molecule which encodes a polypeptide comprising those shown in SEQ ID NO 6 , 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; b) nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,

32, 33, 34, 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45.

A further subject of the present invention is a polypeptide which is encoded by a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of a) nucleic acid molecule which encodes a polypeptide comprising in SEQ ID NO 6, 8, 10, 12, 14, 16 , 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; b) nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34 , 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or

40 coded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) Nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45 includes.

Furthermore, the subject of the present invention is a nucleic acid molecule which codes for a polypeptide which comprises a polypeptide which is encoded by a nucleic acid molecule which differs in one, two, three, four, five, six, seven, eight, nine, ten or more nucleic acids of a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of a) nucleic acid molecule which encodes a polypeptide comprising those shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38 , 39 and / or 40 shown sequence; b) nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34 , 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) Nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45 comprises; and encodes a polypeptide having a hemoprotein activity.

Furthermore, the present invention is a polypeptide activity of a Hemproteins which is encoded by a nucleic acid molecule which differs in one, two, three, four, five, six, seven, eight, nine, ten or more nucleic acids from a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of a) a nucleic acid molecule encoding a polypeptide comprising those shown in SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35 , 36, 37, 38, 39 and / or 40 shown sequence; b) nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34 , 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) Nucleic acid molecule obtained by mixing a nucleic acid molecule from a cDNA database or from a genome database using the primer according to

Sequence Nos. 41 and 42 amplifies f) a nucleic acid molecule encoding a polypeptide having hemoprotein activity which hybridizes under stringent conditions to a nucleic acid molecule according to (a) to (c); and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45; and encodes a polypeptide having a hemoprotein activity.

Another object of the present invention is a DNA expression cassette containing a nucleic acid sequence as described above.

Another object of the present invention is a vector containing an expression cassette containing a nucleic acid sequence as described above.

The present invention furthermore relates to a cell according to the invention, preferably a monocotyledonous organism or a dicotyledonous organism having an increased activity of at least one hemoprotein based on the expression of a nucleic acid sequence as described above.

An object of the present invention is further a cell prepared by the method according to the invention.

In one embodiment of the present invention, by altering the activity of a hemoprotein, in addition to increasing the ATP-ADP ratio, the oil content in the cells increased.

The oil content is the total fatty acid content in the cells according to the invention.

"Oil" includes in the. Meaning of the invention neutral and / or polar lipids and mixtures thereof. By way of example but not by way of limitation, those listed in Table 1 should be mentioned.

Table 1: Herbal lipid classes

Neutral lipids preferably means triacylglycerides. Both neutral and polar lipids can contain a wide range of different fatty acids. By way of example, but not by way of limitation, the fatty acids listed in Table 2 should be mentioned.

Table 2: Overview of different fatty acids (selection)

¹ chain length: number of double bonds ^* not naturally occurring in plants

For further information please refer to the Römpp Chemie Lexikon - CD Version 2.0, Stuttgart / New York: Georg Thieme Verlag 1999.

In a preferred variant, the content of unsaturated fatty acids is increased, in particular to linolenic acid.

By increasing the total oil content of the cell according to the invention, the total content of proteins is not or only slightly reduced. That is, the total content of fatty acids, expressed in mass to mass dry weight, is significantly increased over that of the wild-type. The total protein content compared to that of the wild-type, also expressed in mass by mass dry weight, remains constant or minimally reduced. The reduction is less than the increase in the oil content in percentage, relative to the wild type.

The increase of the ATP-ADP ratio, ie the increase of the energy status by storage of the energy in ATP, remains constant in cells which show an increased activity of hemproteins due to the method according to the invention. This means, the ATP-ADP ratio of the cells is not affected by a change in the external conditions.

For the purposes of the invention, external conditions are defined as the culture conditions for cells, tissues, organs, microorganisms or plants. This can z. For example, composition of the medium, temperature, composition of the atmosphere or other factors influencing the wild-type.

In one embodiment of the present invention, the ATP-ADP ratio of the cells with increased hemoprotein activity according to the invention when lowering the oxygen concentration in the surrounding atmosphere to 4% is at least 200%, 300%, preferably 400%, particularly preferably at least 500% or more related to the ATP-ADP ratio of the wild-type.

In addition, the lactam content formed under these anaerobic culture conditions is at most 80%, preferably 75%, 70%, more preferably 65%, 60%, 55%, 50% or less, based on the amount of wild-type lactate. In a further embodiment of the invention, the change in the hemoprotein activity, the increased ATP-ADP ratio, the increased oil content and / or the reduced amount of lats is a stable feature of the cells of the invention which persists over several generations, preferably up to T2 , especially preferred for T3 generation.

In a preferred variant of the present invention, the cells according to the invention are plant cells, organs, parts of plants or whole plants.

"Plant" in the context of the invention means all dicotyledonous or monocyledonous plants. "Plants" in the context of the invention are plant cells, tissues, organs or whole plants such as seeds, tubers, flowers, pollen, fruits, seedlings, roots, leaves, stems or other parts of plants to understand. In addition, plant propagation material such as seeds, fruits, seedlings, cuttings, tubers, cuts or rhizomes to understand.

Included within the term "plants" are also the mature plants, seeds, shoots and seedlings, as well as derived parts, propagation material, plant organs, tissues, protoplasts, callus and other cultures, for example cell cultures, as well as all other types of groupings of Plant cells to functional or structural units. Mature plants means plants at any stage of development beyond the seedling. Keimling means a young, immature plant at an early stage of development. "Plant" also includes annual and perennial dicotyledonous or monocotyledonous plants and includes, by way of example but not limitation, those of the genera Bromus, Asparagus, Pennisetum, Lolium, Oryza, Zea, Avena, Hordeum, Seeale, Tritiumum, Sorghum and Saccharum one. In a preferred embodiment, the method is applied to monocotyledonous plants, for example from the family Poaceae, more preferably to the genera Oryza, Zea, Avena, Hordeum, Seeale, Triticum, Sorghum, and Saccharum, most preferably to plants of agricultural importance , such as Hordeum vulgare (barley), Triticum aestivum (wheat), Triticum aestivum subsp.spelta (spelled), triticale, Avena sative (oats), sea ale cereale (rye), sorghum bicolor (millet), Zea mays (corn), Saccha rum officinarum (sugarcane) or Oryza sative (rice) applied.

Preferred monocotyledonous plants are in particular selected from monocotyledonous crops, such as, for example, the family of Gramineae such as rice, maize, wheat or other cereals such as barley, millet, rye, triticale or oats, as well as sugarcane and all types of grasses. Particularly preferred from the Gramineae family are rice, maize, wheat and barley.

A transformed plant according to the invention is thus a genetically modified plant.

According to the invention, all plants are suitable for carrying out the method according to the invention. Preference is given to potatoes, Arabidopsis thaliana, rapeseed, soybeans, peanuts, corn, manioc, purging nut, yams, rice, sunflower, rye, barley, hops, oats, hard and common wheat, lupins, peas, clover, beets, cabbage, vines, etc. as they are z. B. from the Ordinance on the List of Seeds to the Seed Traffic Act (sheet for PMZ 1986 p. 3, last modified sheet for PMZ 2002 p. 68) are removed.

1. Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, such as, for example

Asteraceae like Sunflower, Tagetes or Calendula,

- Compositae, especially the genus Lactuca, especially the species sativa

(Salad),

Cruciferae, especially the genus Brassica, in particular the species napus (rapeseed), campestris (turnip), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other types of cabbage; and the genus Arabidopsis, especially the species thaliana and cress or canola,

Cucurbitaceae such as melon, pumpkin or zucchini,

Leguminosae especially the genus Glycine, especially the species Glycine max (soybean) as well as alfalfa, pea, bean plants or peanut.

Rubiaceae, preferably of the subclass Lamiidae such as Coffea arabica or Coffea liberica (coffee shrub),

Solanaceae especially the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanum, especially the species tuberosum (potato) and melongena (eggplant) and tobacco or paprika,

Sterculiaceae, preferably of the subclass Dilleniidae such as Theobroma cacao (cocoa bush),

Theaceae, preferably of the subclass Dilleniidae such as Camellia sinensis or Thea sinensis (tea shrub),

- Umbelliferae, especially the genus Daucus (especially the species carota

(Carrot)) and Apium (especially the species graveolens dulce) and others; and the genus Capsicum, especially the annum species (pepper),

and linseed, soy, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the various tree, nut and wine varieties, especially banana and kiwi.

Also included are ornamental plants, ornamental or ornamental trees, flowers, cut flowers, shrubs or lawns. Exemplary but not restrictive are angiosperms, bryophytes such as Hepaticae (liverwort) and Musci (Moose); Pteridophytes such as ferns, horsetail and lycopene; Gymnosperms such as Conifers, Cycads, Ginkgo and Gnetalen, the Rosaceae families such as Rose, Ericaceae such as Rhododendrons and Azaleas, Euphorbiaceae such as Poinsettias and Cronoton, Caryophyllaceae such as Cloves, Solanaceae such as Petunia, Gesneriaceae such as African Violet, Balsaminaceae such as the Springwort Orchidaceae such as orchids, iridaceae such as gladioli, iris, freesia and crocus, compositae such as calendula, geraniaceae such as geranium, liliaceae such as the dragon tree, moraceae such as ficus, araceae such as philodendron and others. Particularly interesting is the use of oil plants, ie plants that already naturally have a high oil content and / or are used for industrial extraction of oils. These plants may have a high oil content and / or a particular, industrially interesting fatty acid composition. Preference is given to plants which have a lipid content of at least 1% by weight. Oil plants include, by way of example: Bovago oficinalis (borage); Brassica species such as B. campestris, B. napus, B. rapa (mustard or rapeseed); Cannabis sativa (hemp); Curthamus tinctorius (Dyer's Thistle); Cocos nucifera (coconut); Crambe abyssinica (Krambe); Cuphea species (Cuphea species provide fatty acids of medium chain length, especially for industrial applications); Elaeis guinensis (African oil palm); Ekeis oleiferu (American oil palm); Glycine max (soybean); Gossypium hirisfum (American cotton); Gossypium barbadense (Egyptian cotton); Gossypium herbaceous (Asian Cotton Cotton); Helianthus annus (sunflower); Jatropha curcas (purging nut or vomit nut); Linum usitatissimum (flax or flax); Oenethera biennis (Evening primrose); Ozea europea (olive); Oryza sativa (Rice); Ricinus communis (Castor); Sesamum indicum (sesame); Glycine max (soybean); Triticum species (wheat); Zea maize (maize) and various types of nuts such as walnut or almond.

When the plants used are those plants belonging to the genus of legumes, the expression of foreign proteins leghemoglobins or naturally non-symbiotic hemoglobins or alteration of the plants is such that they are natural overexpressing leghemoglobin or non-symbiotic hemoglobin.

Highly preferred are potatoes, Arabidopsis thaliana, rapeseed and soybean.

It is advantageous if the aforementioned plants are a leghemoglobin selected from the group consisting of leghemoglobin from the plants Lupinus luteus (LGB1_LUPLU, LGB2_LUPLU), Glycine max (LGBA_SOYBN, LGB2_SOYBN, LGB3_SOYBN), Medicago sativa (LGB1-4_MEDSA), Medicago trunculata (LGB1_MEDTR ), Phaseolus vulgaris (LGB1_PHAVU, LGB2_PHAVU), Vicia faba

(LGB1_VICFA, LGB2_VICFA), Pisum sativum (LGB1_PEA, LGB2_PEA), Vigna unguiculata (LGB1_VIGUN), Lotus japonicus (LGB_LOTJA), Psophocarpus tetragonolobus (LGB_PSOTE), Sesbania rostrata (LGB1_SESRO), Casuarina glauca (HBPA_CASGL), and Canvalaria lineata (HBP_CANLI express). In brackets, the respective Swiss-Prot database entries are noted. It is particularly advantageous if the abovementioned plants are a non-symbiotic hemoglobin selected from the group consisting of hemoglobin from the plants Arabidopsis thalina (AT_AHB2), Brassica napus (BN_AHB2), Linum usitatissimum (LU_AHB2), Glycine max (GM_AHB2), Helianthus annus (HA_AHB2), Triticum aestivum (TA_AHB2), Hordeum vulgaris (HV_AHB2), Oryza sativa (OS_AHB2) and Zea mays (ZM_AHB2).

Particularly advantageous are plants which have the sequence no. 1 (AT-AHB2) coding for non-symbiotic hemoglobin.

In a preferred variant of the invention are plants expressing the Hemprotein memory organ specifically.

These are z. As bulbs, tubers, seeds, grains, nuts, leaves, etc. Under storage organs in the context of the invention are to be understood as fruits. Fruits are the collective name for the organs of the plants surrounding the seeds as nutrient tissue. Not only edible fruits, especially fruits, but also legumes, cereals, nuts, spices should be considered, as well as officially used drugs (see Fructus, Semen). Of course, a storage of the storage materials in the entire plant can take place.

Preferably, the hemprotein is expressed in a tuber-specific or seed-specific manner.

As plants, all of the previously mentioned come into consideration. It is particularly preferred if it is tuber-producing plants, in particular potato plants or seed-producing plants, in particular Arabidopsis thaliana or oilseed rape.

The tissue-specific expression can, for. B. be achieved by using a tissue-specific promoter. Such a tissue-specific expression is known, for example, from US Pat. No. 6,372,961 B1, column 11, lines 44 ff.

In a further embodiment, the present invention relates to the use of the above-described nucleic acid molecules coding for polypeptides with the activity of hemoproteins for the production of cell, tissue, organ, microorganism or plant with increased ATP-ADP ratio and / or altered oil content, preferably increased fatty acid content, preferably increased linolenic acid content The invention will be described by way of example with reference to the following experiments.

Examples

General methods:

All chemicals, unless otherwise mentioned, come from the companies Fluka (Buchs), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Restriction enzymes, DNA modifying enzymes, and molecular biology kits were purchased from Amersham-Pharmacia (Freiburg), Biometra (Göttingen), Roche (Mannheim), New England Biolabs (Schwalbach), Novagen (Madison, Wisconsin, LISA), Perkin Elmer (Weiterstadt), Qiagen (Hilden), Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion (Cambridgeshire, United Kingdom). The reagents used were used according to the manufacturer's instructions.

The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The carried out in the context of the present invention cloning steps such. B. restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, growth of bacteria, propagation of phages and sequence analysis of recombinant DNA are as in Sambrook et al , (1989) CoId Spring Harbor Laboratory Press; ISBN 0-87969-309-6 described. The sequencing of recombinant DNA molecules is carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74: 5463-5 547).

Example 1: Cloning of Arabidopsis thaliana genes AHB1 and AHB2

To clone the AHB2 gene, total RNA was extracted from 6 week old Arabidopsis plants. The preparation of the corresponding cDNA was carried out by RT-PCR using the SUPERSCRIPT II (Invitrogen).

For the cloning of the AHB2 gene, the isolated Arabidopsis cDNA was used in a PCR reaction with the oligonucleotide primers AHb2f and AHb2r.

Sequence primer Ahb2f SEQU.ID.No: δ'-TTTGGTACCATGGGEGAGATTGGGTTTACAGAG-S ' Sequence primer Ahb2r SEQU.ID.No: δ'-TTTGGATCCTTATGACCTTTCTTGTTTCATCTCGG-S '

Composition of the PCR approach (50 μL):

5.00 μl of cDNA from Arabidopsis thaliana

5.00 μL 10x buffer (Advantage polymerase) + 25 mM MgCl ₂

5.00 μL 2mM dNTP

1.25 μL per primer (10 pmol / μL)

0.50 μL Advantage polymerase

The Advantage polymerase from Clontech was used.

PCR program:

Initial denaturation for 2 min at 95 ° C, then 35 cycles at 45 sec 95 ° C, 45 sec 55 ° C and 2 min 72 ° C. Final extension of 5 min at 72 ° C.

The PC R preparations were then separated by agarose gel electrophoresis and the amplified DNA fragments of AHB2 were excised from the gel and purified with Qiagen's gel gelification kit according to the manufacturer's instructions and eluted with 50 μl elution buffer.

Subsequently, the DNA fragments were cloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions, resulting in the vector pCR2.1-AHB2 and the sequence was checked by sequencing.

Subsequently, the coding sequences for AHB2 were cloned into a binary plant vector, such as pBIN, behind the seed-specific USP promoter (Bäumein et al. (1991) Mol Gen Genet 225 (3): 459-467). For this purpose, the vector pCR2.1-AHB2 was digested with the restriction enzymes Kpnl and BamHI. The resulting DNA fragments were separated by agarose gel electrophoresis and the AHB coding fragments were excised from the gel and purified with the. Gelpurification'Kit from Qiagen according to the manufacturer and eluted with 50 .mu.L elution buffer. The eluted DNA fragments were ligated with the binary enzyme digested with the same enzymes overnight at 16 ° C (T4 ligase from New England Biolabs). The ligation products are then transformed into TOP10 cells (Stratagene) according to the manufacturer's instructions and selected accordingly. Positive clones are verified with the primers AHb2f and AHb2r by PCR and sequencing. Example 3: Transformation of Agrobacterium

The Agrobacterium -mediated plant transformation can be carried out, for example, using the Agrobacterium tumefaciens strains GV3101 (pMP90) (Koncz and Schell (1986) Mol Gen Genet 204: 383-396) or LBA4404 (Clontech). The transformation can be performed by standard transformation techniques (Deblaere et al., (1984) Nucl Acids Res 13: 4777-4788).

Example 4: Plant transformation

The Agrobacterium -mediated transformation of Arabidopsis thaliana was carried out using standard transformation and regeneration techniques (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ. 1995, in Sect, Ringbuc Central Signature: BT1 1-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 p., ISBN 0-8493-5164-2). The use of antibiotics for the Agrobacterium and

Plant selection depends on the binary vector and Agrobacterium strain used for the transformation. The selection of AHB2-transformed Arabidopsis thaliana plants was carried out with hygormycin.

The Agrobacterium -mediated transformation of oilseed rape can e.g. By cotyledon or hypocotyl transformation (Moloney et al., Plant Cell Report 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 694-701). The use of antibiotics for Agrobacterium and plant selection depends on the binary vector and Agrobacterium strain used for the transformation.

The Agrobacterium-mediated gene transfer in flax (Linum usitatissimum) can be determined using, for example, one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285.

Transformation of soy may be accomplished using, for example, a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 039 7 687, US 5,376,543, US 5,169,770 (University Toledo).

Plant transformation using particle bombardment,

Polyethylene glycol-mediated DNA uptake or via the silicon carbonate fiber technique is described for example by Freeling and Walbot "The maize handbook" (1993) ISBN 3-540-97826-7, Springer publishing house New York).

Example 5: Examination of the Expression of a Recombinant Gene Product in a Transformed Organism

A suitable method for determining the amount of transcription of the gene (an indication of the amount of RNA available for translation of the gene product) is the performance of a Northern blot as set forth below (for reference, see Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley: New York, or the example part above), wherein a primer designed to bind to the gene of interest is labeled with a detectable label (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, separated on a gel, transferred to a stable matrix and incubated with this probe, the binding and extent of binding of the probe is the presence as well as the amount of mRNA for this Gene indicates. This information indicates the degree of transcription of the transformed gene. Total cellular RNA may be prepared from cells, tissues or organs by a variety of methods, all known in the art, such as that described by Bormann, E.R., et al. (1992) Mol. Microbiol. 6: 317-326.

Northern hybridization:

For RNA hybridization, total RNA was extracted from maturing seeds using the Concert RNA Plant Reagent (Invitrogen GmbH, Karlsruhe, Germany). 20 μg total RNA or 1 μg poly (A) + RNA was separated by gel electrophoresis in 1.25% strength agarose gels using formaldehyde as described in Amasino (1986, Anal. Biochem. 152, 304), by capillary attraction using 10 x SSC on positively charged nylon membranes (Hybond N +, Amersham, Braunschweig), immobilized by UV light and incubated for 3 hours at 68 ° C using hybridization buffer (10% dextran sulfate, w / o, 1 M NaCl, 1% SDS, 100 mg herring sperm DNA). The labeling of the DNA probe with the

High Prime DNA labeling kit (Roche, Mannheim, Germany) during the prehybridization step, using alpha- ³² P-dCTP (Amersham Pharmacia, Braunschweig, Germany). Hybridization was performed after addition of the labeled DNA probe in the same buffer at 68 ° C overnight. The washing steps were performed twice for 15 min using 2 X SSC and twice for 30 min using 1 X SSC, 1% SDS, at 68 ° C. Exposure of the sealed filters was performed at -70 ° C for a period of 1 to 14T.

Standard techniques, such as western blotting, can be used to study the presence or relative amount of protein translated from this mRNA (see, for example, Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley: New York). In this method, the total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe such as an antibody that specifically binds to the desired protein. This probe is usually provided with a chemiluminescent or colorimetric label which is easily detected. The presence and amount of label observed indicates the presence and amount of the desired protein present in the cell.

Figure 1 shows the results of the Northern blot of 3 independent transgenic Arabidopsis lines transformed with the AHB2 construct as well as the

Wild type. The plants of lines 9, 10 and 11 show a strong detection signal on the Northern blot. The plants consequently express the AHB2 gene in ripening seeds. By contrast, only a weak signal based on the expression of the endogenous AHB2 gene could be detected in the semen sample of the wild-type.

Example 6: Analysis of the effect of the recombinant proteins on the production of the desired product

The effect of genetic modification in plants or on the production of a desired compound (such as a fatty acid) can be determined by culturing the modified plant under appropriate conditions (such as those described above) and increasing the medium and / or cellular components Production of the desired product (ie of lipids or a fatty acid) is examined. These analytical techniques are well known to those skilled in the art and include spectroscopy, thin layer chromatography, staining methods of various types, enzymatic and microbiological methods, and analytical

Chromatography such as high performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987 ) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, Chapter IM: "Product Recovery and Purification", pp. 469-714, VCH: Weinheim; Belter, PA, et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, JF, and Cabral, JMS (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, JA, and Henry, JD (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 1 1, pp. 1 -27, VCH: Weinheim; and Dechow, FJ. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to the above-mentioned methods, plant lipids derived from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Be. USA 96 (22): 12935-12940, and Browse et al. (1986) Analytic Biochemistry 152: 141-145. Qualitative and quantitative lipid or fatty acid analysis is described in Christie, William W.

Advances in Lipid Methodology, Ayr / Scotland: OiIy Press (OiIy Press Lipid Library; 2);

Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland: OiIy Press, 1989, Repr. 1992, IX, 307 p. (OiIy Press Lipid Library, 1);

"Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) - 16 (1977) et al.

Progress in the Chemistry of Fats and Other Lipids CODES.

One example is the analysis of fatty acids (abbreviations: FAME, fatty acid methyl ester, GC-MS, gas-liquid chromatography-mass spectrometry, TAG, triacylglycerol, TLC, thin-layer chromatography).

The unambiguous evidence for the presence of fatty acid products can be obtained by analysis of recombinant organisms by standard analytical methods: GC, GC-MS or TLC as variously described by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Edition. Christie, Oliver Press, Dundee, 1 19-169, 1998, Gas Chromatography Mass Spectrometry Method, Lipids 33: 343-353).

The material to be analyzed may be broken up by sonication, milling in the glass mill, liquid nitrogen and milling or other applicable methods. The material must be centrifuged after rupture. The sediment is distilled in aqua. re-suspended, heated at 100 ° C for 10 min, cooled on ice and recentrifuged, followed by extraction into 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 h at 90 ° C resulting in hydrolyzed oil and lipid compounds. which give transmethylated lipids. These fatty acid methyl esters are extracted in petroleum ether and finally added to a GC. Subjected to analysis using a capillary column (Chrompack, WCOT fused silica, CP-Wax-52 CB, 25 microm, 0.32 mm) at a temperature gradient between 170 ° C and 240 ° C for 20 min and 5 min at 240 ° C. The identity of the resulting fatty acid methyl esters must be defined using standards available from commercial sources (ie Sigma).

Plant material is first mechanically homogenized by mortars to make it more accessible to extraction.

The following protocol was used for quantitative and qualitative oil analysis of Arabidopsis plants transformed with the constructs ADH1 and ADH2:

The extraction of the lipids from seeds is carried out according to the method of Bligh & Dyer (1959) Can J Biochem Physiol 37:91 1. For this purpose, 5 mg Arabidopsis Brassica seeds are weighed in 1.2 ml Qiagen microtubes (Qiagen, Hilden) on a Sartorius (Göttingen) microbalance. The seed material is homogenized with 1 ml of chloroform / methanol (1: 1, Sigma's mono-C15-glycerin as internal standard) in the Rätschmühle MM300 from Retsch (Haan) and incubated for 20 min at RT. After centrifugation, the supernatant was transferred to a new vessel and the sediment extracted again with 1 ml of chloroform / methanol (1: 1). The supernatants were combined and concentrated to dryness. The derivatization of the fatty acid was carried out by acid methanolysis. For this purpose, the extracted lipids were treated with 0.5 M sulfuric acid in methanol and 2% (v / v) dimethoxypropane and for 60 min. incubated at 80 ° C. This was followed by extracting twice with petroleum ether, followed by washing steps with 100 mM sodium bicarbonate and water. The fatty acid methyl esters thus prepared were concentrated to dryness and taken up in a defined volume of petroleum ether. 2 μl of the fatty acid methyl ester solution were finally separated by gas chromatography (HP 6890, Agilent Technologies) on a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and analyzed by means of a flame ionization detector.

The quantification of the oil was carried out by comparing the signal strengths of the derivatized fatty acids with those of the internal standard mono-C15-glycerol (Sigma).

The determination of the fatty acid profile was made by relative comparison of the signal strengths to each other. The unsaturation / saturation index (USI) was determined as described in Gutierrez et al. ((2005) Food Chemistry 90, 341-346) and reflects the ratio of unsaturated to saturated fatty acids in the Seed oil.

The quantitative protein analysis of the Arabidopsis plants transformed with the construct USP-AHB2 was applied to the protocol of Bradford (1976). Bovine serum albumin served as standard.

Table 3: Oil content (total fatty acid content) in mature and maturing (13-14 DAF) seeds of transgenic Arabidopsis lines transformed with the USP AHB2 construct and in mature and ripening (13-14 DAF) seeds of untransformed wild-type plants. In mature seeds, the oil content was determined over three consecutive generations. The listed values correspond to mean values and standard errors from 6 independent measurements. Significant changes to the wild-type (based on the statistical t-test analysis, p <0.05) are highlighted by asterisks ( ^* ).

Table 3 shows, almost by way of example, the course of the oil pastes in mature seeds of 3 independent transgenic Arabidopsis lines over 3 generations transformed with the construct USP-AHB2 and the untransformed wild-type plants. The values correspond to the mean values from 6 independent measurements. The standard errors are also indicated. Significant changes to the wild type (based on the statistical t-test analysis) are highlighted by asterisks ( ^* ). In all 3 generations could in the mature seeds of the transgenic Lines are shown a significant increase in oil content. The phenotype achieved is accordingly stable over several generations. In addition, a significantly higher oil content in the transgenic lines was also found in maturing T3 seeds during the oil storage phase (see Table 1).

Figure 2 shows, by way of example, the results for the quantitative determination of the oil and protein contents in T3 seeds of 3 independent transgenic Arabidopsis lines (9, 10, 11) which had been transformed with the construct USP-AHB2 and in the seeds of the untransformed wild-type Plants. The values correspond to the mean values from 10 independent measurements. The standard errors are also indicated. Significant changes to the wild type (based on the statistical t-test analysis) are highlighted by asterisks ( ^* ). In all 3 transgenic lines, a significant increase in oil content by 15% (line 9), 31% line (line 10) and 40% (line 1 1). The different oil increases in the different lines correlate with the expression levels shown in Figure 2. In contrast, overexpression of AHB2 has no effect on the oil content

FIG. 3 shows, by way of example, the results of the qualitative oil analysis in the mature seeds of transgenic Arabidopsis lines which had been transformed with the construct USP-AHB2 and in the seeds of the untransformed wild-type plants (A. linoleic acid content, B. linolenic acid content , C. linoleic acid / linolenic acid ratio and D. USI (unsaturation / saturation index)). The values correspond to the mean and standard errors from 10 independent measurements. Significant changes to the wild type (based on the statistical t-test analysis) are highlighted by asterisks ( ^* ). Semen-specific overexpression of AHB2 in seed oil leads to a significant increase of alpha-linolenic acid (C18: 3) from 25% in the wild-type plant to more than 30% in transgenic lines 10 and 11. The content of linoleic acid (C18: 2) precursor of C18 : 3 is unchanged. This is also reflected in the ratio of C18: 3 to C18: 2 (0.8 in the seed oil of the wild-type plants and> 1 in the seed oil of the transgenic plants.) Overexpression of AHB2 consequently leads to an increased de-saturation of the fatty acids in the Seeds of the transgenic lines are also reproduced by the USI, which increases from 9 in the wild-type seeds up to 12 in the transgenic seeds.

Example 7: Determination of the ATD / ADP ratio and the lactic acid content. In order to investigate the influence of different oxygen concentrations on the metabolism in the seeds of wild-type and AHB2-overexpressing Arabidopsis plants, the plants were grown in a greenhouse (21 ° C / day and 17 ° C / night, 50% humidity day and night, photoperiod 16 h day / 8 h night, night intensity 180 μmol photons nτ ² s ^"1 ) For the incubation experiments with different concentrations of oxygen, puffed stems were packed in a transparent plastic bag in the air with an oxygen content of 21% and 4% (v / v The air mixtures from Messer Griesheim GmbH (Magdeburg, Germany) contained 350 ppm CO2, oxygen concentrations as indicated above and nitrogen After 2 hours, the pods were harvested and flash frozen immediately in liquid nitrogen Seeds were lyophilized from 13-14 days old Prepared pods as described in Gibon et al. (2002) Plant J 30: 221-235.

To analyze the metabolites ATP, ADP and lactic acid, seeds were homogenized in a liquid nitrogen-cooled vibrating mill made by Retch (Haan, Germany) and then subjected to extraction with trichloroacetic acid. The quantification of the metabolites was subsequently carried out in Gibon et al. (2002) Plant J 30: 221-235.

Figure 4 shows the effect of seed specific expression of AHB2 on the ATP / ADP ratio (A) and lactic acid content (B) in ripening seeds cultured under normal (21%) oxygen conditions or under hyoxic conditions (4%). The results correspond to mean and standard errors from 6 independent measurements. Significant changes to the wild type (based on the statistical t-test analysis) are highlighted by asterisks ( ^* ).

The seed-specific overexpression of AHB2 leads to a 2-4-fold higher ATP to ADP ratio in the seeds of the transgenic lines (4-8) than in the wild-type seeds under natural oxygen concentrations in the environment (2). This indicates an improved energy supply through the respiratory chain in the transgenic seeds even under the low oxygen concentrations within the seed.

Lowering the oxygen concentration in the environment to 4% leads to a reduction of the energy status in the wildtype seeds, which is reflected in the reduction of the ATP to ADP ratio from 2 to 0.4. The lowering of the energy status was associated with an accumulation of lactic acid in the seeds (20 μmol gDW " ¹ at 21% O2, 50 μmol gDW" ¹ ). This demonstrates that the wild-type seeds partially compensate for missing energy by energetically less favorable, anaerobic fermentation. Lowering the oxygen concentration in the environment to 4% also leads to a reduction in energy status in the seeds over-expressing AHB2. However, the ATP to ADP ratio in these plants is 0.8 - 2, which is significantly higher than in the wild-type seeds (0.4). This indicates a still adequate aerobic energy supply with an oxygen concentration in the environment of 4%. This is confirmed by the fact that in the transgenic seeds does not increase the lactic acid formed by aerobic fermentation.

Claims

claims

1. A method for altering the ATP-ADP ratio in at least one cell, tissue, organ, microorganism or plant, characterized in that the activity of at least one Hemproteins is changed.

2. The method according to claim 1, characterized in that the activity of at least one leghemoglobin is changed.

3. The method according to any one of claims 1 or 2, characterized in that the activity of a Hemproteins is increased.

4. The method according to any one of claims 1 to 3, characterized in that in the activity of at least one polypeptide is increased, which is encoded by a nucleic acid molecule comprising at least one nucleic acid molecule selected from the group consisting of: a) nucleic acid molecule encoding a polypeptide comprising in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or

40 sequence shown; b) A nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34 , 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule coding for an inhibitory protein which is prepared from a DNA library using a nucleic acid molecule according to (a) to (c) or Partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45.

5. The method according to any one of claims 1 to 4, characterized in that in the activity of at least one Hemproteins by expression, preferably over expression, which is encoded by a nucleic acid molecule comprising at least one nucleic acid molecule selected from the group consisting of: a) nucleic acid molecule, the a polypeptide comprising the amino acid sequence shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 sequence shown; b) A nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29 30 and / or 31; c) a nucleic acid molecule which codes for a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14,

16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) nucleic acid molecule coding for a polypeptide comprising an amino acid sequence according to the consensus sequence of the hemprotein sequences comprising SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, more preferably SEQ ID NO 43 and / or 45; is increased

6. The method according to any one of claims 1 to 5, characterized in that the leghemoglobin and hemoglobin from plants from the group consisting of Arabidopsis thaliana, Lupinus luteus, Glycine max, Medicago sativa, Medicago trunculata, Phaseolus vulgaris, Vicia faba, Pisum sativum, Vigna unguiculata, Lotus japonicus, Psophocarpus tetragonolobus, Sesbania rostrata, Casarina glauca and Canvalaria lineata.

7. The method according to any one of claims 1 to 6, characterized in that the hemoprotein is derived from lotus japonicus or preferably Arabidopsis thaliana.

8. The method according to any one of claims 1 to 7, characterized in that the plants are transformed in such a way that it expresses the hemoprotein gene specific for storage.

9. The method according to any one of claims 1 to 8, characterized in that the plants are transformed such that they expressed the Hemprotein tuber-specific and / or seed-specific.

10. The method according to any one of claims 1 to 9, characterized in that monocotyledonous crops, in particular of the species Gramineae be transformed.

1 1. A method according to any one of the preceding claims 1 to 10, characterized in that dicotyledonous crops, in particular the family

Asteraceae, Brassicacea, Compositae, Cruciferae, Cucurbitaceae, Leguminose, Rubiaceae, Solanaceae, Sterculiaceae, Theaceae or Umbelliferae.

12. The method according to any one of the preceding claims 1 to 1 1, character- ized in that potatoes, Arabidopsis thaliana, soybean or oilseed rape are transformed.

13. Use of a nucleic acid molecule comprising at least one nucleic acid molecule selected from the group consisting of: a) nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18 , 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; b) A nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29 30 and / or 31; c) a nucleic acid molecule which codes for a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14,

16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) Nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45 comprises; for producing a polypeptide having hemoprotein activity in at least one

Cell, tissue, organ, microorganism or plant.

14. Use of a nucleic acid molecule comprising at least one nucleic acid molecule selected from the group consisting of: a) nucleic acid molecule which encodes a polypeptide, comprising those shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 , 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or

40 sequence shown; b) A nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29 30 and / or 31; c) a nucleic acid molecule which codes for a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14,

16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) Nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45 comprises; for altering the ATP-ADP ratio in at least one cell, tissue, organ, microorganism or plant.

15. Use of a nucleic acid molecule comprising at least one nucleic acid molecule selected from the group consisting of: a) nucleic acid molecule which encodes a polypeptide, comprising those shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 , 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or

40 sequence shown; b) A nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) Nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45 comprises; for producing at least one cell, tissue, organ, microorganism or

Plant with altered ATP-ADP ratio, preferably increased ATP-ADP ratio.

16. Use of a nucleic acid molecule comprising at least one

A nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule which encodes a polypeptide, comprising those shown in SEQ ID

NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 shown sequence; b) A nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34 , 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37,

38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45; for producing at least one cell, tissue, organ, microorganism or plant with altered oil content, preferably increased fatty acid content, preferably increased linolenic acid content.

17. A nucleic acid molecule which codes for a polypeptide which comprises a polypeptide which is encoded by a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of a) nucleic acid molecule which encodes a polypeptide, comprising those shown in SEQ ID NO 6, 8, 10 , 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; b) nucleic acid molecule comprising at least one polynucleotide of the sequence shown in SEQ ID Nos. 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31; c) a nucleic acid molecule which encodes a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34 , 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to sequence Nos. 41 and 42 f) a nucleic acid molecule which codes for a polypeptide with hemoprotein activity which hybridizes under stringent conditions with a nucleic acid molecule according to (a) to (c); and g) a nucleic acid molecule encoding a hemoprotein which consists of a DNA library using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt can be isolated as a probe under stringent hybridization conditions; h) nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45.

A nucleic acid molecule encoding a polypeptide comprising a polypeptide encoded by a nucleic acid molecule that differs in one, two, three, four, five, six, seven, eight, nine, ten or more nucleic acids from a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of a) nucleic acid molecule which encodes a polypeptide, comprising those shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 shown sequence; b) nucleic acid molecule which comprises at least one polynucleotide of the sequence shown in SEQ ID NO 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30 and / or 31; c) a nucleic acid molecule which codes for a polypeptide whose sequence has an identity of at least 40% to the sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14,

16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40; d) Nucleic acid molecule according to (a) to (c) which is suitable for a fragment of the sequences according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 33, 34, 35, 36, 37, 38, 39 and / or 40 encoded; e) nucleic acid molecule which is obtained by amplifying a nucleic acid molecule from a cDNA database or from a genome database using the primers according to Sequence Nos. 41 and 42 f) nucleic acid molecule which encodes a polypeptide with hemoprotein activity which under stringent conditions with a Nucleic acid molecule according to (a) to (c) hybridized; and g) nucleic acid molecule coding for a hemprotein which consists of a DNA Bank can be isolated using a nucleic acid molecule according to (a) to (c) or its partial fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt as a probe under stringent hybridization conditions; h) Nucleic acid molecule coding for a polypeptide containing an amino acid sequence according to the consensus sequence of the hemprotein sequences, SEQ ID NO 46 and / or 47, preferably SEQ ID NO 43 and / or 44, particularly preferably SEQ ID NO 43 and / or 45 comprises; and encodes a polypeptide having a hemoprotein activity.

19. Protein encoded by the nucleic acid molecule according to claim 17 or 18, wherein the protein does not consist of the amino acid sequence shown in SEQ ID. NO. 2 and 4 sequence shown.

20. A DNA expression cassette comprising a nucleic acid sequence which is substantially identical to a nucleic acid molecule according to claim 17 or 18 and encoded for a protein according to claim 19.

21. A vector containing an expression cassette according to claim 20.

22. Transgenic cell containing an expression cassette according to claim 20 or a vector according to claim 21.