EP2007894A2 - Procédé de modification du rapport atp/adp dans des cellules - Google Patents

Procédé de modification du rapport atp/adp dans des cellules

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Publication number
EP2007894A2
EP2007894A2 EP07727132A EP07727132A EP2007894A2 EP 2007894 A2 EP2007894 A2 EP 2007894A2 EP 07727132 A EP07727132 A EP 07727132A EP 07727132 A EP07727132 A EP 07727132A EP 2007894 A2 EP2007894 A2 EP 2007894A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
acid molecule
seq
polypeptide
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07727132A
Other languages
German (de)
English (en)
Inventor
Thorsten Zank
Oliver Oswald
Jörg BAUER
Helene Vigeolas
Peter Geigenberger
Mark Stitt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Institut fuer Molekulare Pflanzenphysiologie
Original Assignee
Max Planck Institut fuer Molekulare Pflanzenphysiologie
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Publication date
Application filed by Max Planck Institut fuer Molekulare Pflanzenphysiologie filed Critical Max Planck Institut fuer Molekulare Pflanzenphysiologie
Priority to EP07727132A priority Critical patent/EP2007894A2/fr
Publication of EP2007894A2 publication Critical patent/EP2007894A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins

Definitions

  • 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 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.
  • hydrolysis of ATP to ADP serves as a driving force in many biochemical processes such.
  • 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.
  • ATP plays a key role in the metabolism of the cell.
  • 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.
  • the intracellular ATP reserves are consumed and the ATP-ADP ratio is shifted towards ADP.
  • hemoglobin or related proteins 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.
  • 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.
  • leghemoglobin in plant cells is also known from Barata et al .: (Plant Science, Vol.155, June 2000, 193-202), wherein oxygen availability is examined.
  • 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.
  • 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-Octadecatrien Acid (ice, ice, cis-9,12,15-Octadecatrien Acid (ice, ice, cis-9,12,15-Octadecatrienklad)
  • the ATP-ADP ratio is meant the ratio of the concentration of ATP to the concentration of ADP.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • RNA ribonucleic acids
  • DNA deoxyribonucleic acids
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • homologs of the nucleic acid molecules of the invention are used.
  • Gap Weight 50 Length Weight: 3
  • SEQ ID NO: 1, 3, 5 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
  • 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.
  • oligonucleotides 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.
  • 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.
  • 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.
  • 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.
  • "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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the coding regions (structural genes) preceding (5 " upstream or upstream) and / or subsequent (3 " or downstream) sequences are included.
  • this includes sequence regions with regulatory function. They can influence transcription, RNA stability or RNA processing as well as translation.
  • 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,
  • 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.
  • 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 S
  • 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 46 and
  • 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,
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the content of unsaturated fatty acids is increased, in particular to linolenic acid.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • plant propagation material such as seeds, fruits, seedlings, cuttings, tubers, cuts or rhizomes to understand.
  • 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.
  • 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.
  • 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 a
  • 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.
  • the family of Gramineae such as rice, maize, wheat and barley.
  • a transformed plant according to the invention is thus a genetically modified plant.
  • 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.
  • Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, such as, for example
  • 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),
  • angiosperms 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,
  • 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);
  • leghemoglobins or naturally non-symbiotic hemoglobins or alteration of the plants is such that they are natural overexpressing leghemoglobin or non-symbiotic hemoglobin.
  • 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
  • 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).
  • AZA_AHB2 Arabidopsis thalina
  • BN_AHB2 Brassica napus
  • L_AHB2 Linum usitatissimum
  • Glycine max Glycine max
  • HA_AHB2 Helianthus annus
  • TA_AHB2 Triticum aestivum
  • HV_AHB2 Hordeum vulgaris
  • plants which have the sequence no. 1 (AT-AHB2) coding for non-symbiotic hemoglobin.
  • plants expressing the Hemprotein memory organ specifically are plants expressing the Hemprotein memory organ specifically.
  • the hemprotein is expressed in a tuber-specific or seed-specific manner.
  • 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.
  • a tissue-specific expression is known, for example, from US Pat. No. 6,372,961 B1, column 11, lines 44 ff.
  • 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.
  • 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).
  • the isolated Arabidopsis cDNA was used in a PCR reaction with the oligonucleotide primers AHb2f and AHb2r.
  • 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 ⁇ l elution buffer.
  • 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.
  • 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).
  • a binary plant vector such as pBIN
  • 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 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).
  • 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).
  • 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 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).
  • a suitable method for determining the amount of transcription of the gene 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.
  • RNA hybridization total RNA was extracted from maturing seeds using the Concert RNA Plant Reagent (Invitrogen GmbH, Düsseldorf, Germany). 20 ⁇ g total RNA or 1 ⁇ g poly (A) + RNA was separated by gel electrophoresis in 1.25% strength agarose gels using formaldehyde as described in Amasino (1986, Anal. Biochem.
  • 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).
  • 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
  • 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 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.
  • a desired compound such as a fatty acid
  • 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
  • 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.
  • Plant material is first mechanically homogenized by mortars to make it more accessible to extraction.
  • 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.
  • 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 Röschmühle MM300 from Retsch (Haan) and incubated for 20 min at RT.
  • 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.
  • 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.
  • 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 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.
  • 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 are highlighted by asterisks ( * ).
  • asterisks * 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.
  • 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.
  • 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 ⁇ mol photons n ⁇ 2 s "1 )
  • 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.
  • 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.

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Abstract

L'invention concerne un procédé de modification du rapport ATP/ADP dans une cellule, un tissu, un organe, un micro-organisme ou une plante par modification de l'activité d'une protéine à hème dans la cellule. L'invention porte également sur l'application de ce procédé.
EP07727132A 2006-04-13 2007-03-20 Procédé de modification du rapport atp/adp dans des cellules Withdrawn EP2007894A2 (fr)

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PCT/EP2007/052655 WO2007118751A2 (fr) 2006-04-13 2007-03-20 Procédé de modification du rapport atp/adp dans des cellules
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DE102006043767A1 (de) * 2006-09-13 2008-03-27 Henkel Kgaa Extrakt aus Apium graveleons zur Stimulierung des Haarwuchses
CN105724726A (zh) 2011-07-12 2016-07-06 非凡食品有限公司 用于消费品的方法和组合物
US20140220217A1 (en) 2011-07-12 2014-08-07 Maraxi, Inc. Method and compositions for consumables
PL2731451T3 (pl) 2011-07-12 2019-03-29 Impossible Foods Inc. Sposoby i kompozycje dla produktów żywnościowych
US10039306B2 (en) 2012-03-16 2018-08-07 Impossible Foods Inc. Methods and compositions for consumables
BR112015016651A2 (pt) 2013-01-11 2017-07-11 Impossible Foods Inc réplica de queijo não láctea compreendendo um coacervado
PT2943078T (pt) 2013-01-11 2021-06-16 Impossible Foods Inc Métodos e composições para consumíveis
SI3044320T1 (sl) * 2013-09-11 2020-07-31 Impossible Foods Inc. Sekrecija polipeptidov, ki vsebujejo hem
JP6759103B2 (ja) 2014-03-31 2020-09-23 インポッシブル フーズ インコーポレイテッド ひき肉レプリカ
EP3959311A1 (fr) 2019-04-25 2022-03-02 Impossible Foods Inc. Compositions et procédés pour la production de protéines contenant de l'hème

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US6372961B1 (en) * 1998-08-20 2002-04-16 Pioneer Hi-Bred International, Inc. Hemoglobin genes and their use
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