EP1506299A2 - Method for producing a transgenic plant having modified transport of substances - Google Patents

Method for producing a transgenic plant having modified transport of substances

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Publication number
EP1506299A2
EP1506299A2 EP03724887A EP03724887A EP1506299A2 EP 1506299 A2 EP1506299 A2 EP 1506299A2 EP 03724887 A EP03724887 A EP 03724887A EP 03724887 A EP03724887 A EP 03724887A EP 1506299 A2 EP1506299 A2 EP 1506299A2
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Prior art keywords
nucleotide sequence
seq
sequence
dna
rna
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German (de)
French (fr)
Inventor
Wolf-Bernd Frommer
Mechthild Tegeder
Axel Hirner
Wolfgang Koch
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • 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
    • C12N15/8245—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 carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • the invention relates to a method for producing a transgenic plant in which the ability to store undesired nitrogen compounds, in particular amino acids, in the harvesting and / or reproductive organs is reduced compared to the wild type.
  • the invention further relates to DNA sequences and RNA sequences corresponding to them, which contain the coding region for an amino acid transporter or parts thereof, their use and vectors, mobile genetic elements, bacteria, host cells, plant cells, plants, seeds and other plant propagation material, which contain the DNA and / or RNA sequences.
  • Plants absorb nitrogen, which is required to build up cell substance, mainly in the form of nitrate, but also as ammonium and, to a lesser extent, as amino acids, for example via the roots.
  • the nitrate is reduced to amino nitrogen and incorporated into organic compounds. This mainly happens in the leaves of the plant.
  • Nitrogen-containing organic compounds, for example amino acids are transported via a vascular system, the phloe, from places of formation such as the leaves to consumption tissues and organs, for example reproductive organs or storage organs (roots, tubers, beets, etc.).
  • the transport processes are particularly important for the shifting of nitrogen compounds in the course of the aging (senescence) of leaves, for example towards the end of the growing season. This removes nitrogen compounds from the dying leaves into other organs, for example Storage organs, relocated to minimize the loss of cell material.
  • AAPs not only mediate the transport of a broad spectrum of amino acids with comparatively low selectivity, but also of other organic nitrogen compounds, for example amides such as glutamine and asparagine as well as citrulline, gamma-aminobutyric acid or auxin. They therefore presumably play a key role in the distribution of organic nitrogen compounds within the plant.
  • Amino acid transporter genes from Arabidopsis thaliana are known for example from EP 0652955.
  • the storage of organic nitrogen compounds such as proteins and amino acids in crop organs of plants is often undesirable.
  • the object of the present invention is therefore to create a possibility of producing plants in which the incorporation of organic nitrogen compounds in their harvesting organs is reduced compared to the wild type.
  • a method which comprises the steps of introducing at least one DNA sequence and / or an RNA sequence corresponding to the DNA sequence and / or a mixed sequence composed of DNA and RNA nucleotides with the coding sequence Region for an amino acid transporter or parts thereof in a plant cell, the DNA and / or RNA sequence and / or mixed sequence in sense or antisense orientation is used and the expression of an endogenous amino acid transporter gene is prevented or reduced, and regenerating a plant from this plant cell, wherein the DNA and / or RNA sequence is a sequence from Beta vulgaris.
  • Plants which have been produced by the process according to the invention have a significantly reduced content of organic nitrogen compounds, in particular amino acids, in their harvesting organs.
  • a plant cell into which the DNA sequence and / or the RNA sequence corresponding to the DNA sequence and / or the RNA / DNA mixed sequence corresponding to the DNA sequence has been introduced in the antisense orientation, the translation of the mRNA of the endogenous amino acid transporter gene hindered by the attachment of an antisense RNA to the RNA.
  • the antisense RNA is formed by transcription of the DNA sequence. In the case of the RNA sequence corresponding to the DNA sequence, this can itself represent the antisense RNA.
  • Post-transcriptional muting post-transcriptional gene silencing
  • PGS post-transcriptional gene silencing
  • RNA interference in that the DNA sequence or an RNA sequence corresponding to the DNA sequence is sense-oriented into the plant cell is introduced, the RNA being used as double-stranded RNA in the case of RNA interference.
  • mixed sequences composed of RNA and DNA sequences chimeric oligonucleotides, as described, for example, by Rice et al. (2000), Plant Physiology 123, 427-437, can be used to mute the endogenous amino transporter gene.
  • the plants produced by the process according to the invention are largely prevented in this way from forming amino acid transporters.
  • tissue-specific promoters By coupling to suitable tissue-specific promoters, it is also possible to specifically target the formation of amino acid transporters in certain tissues or organs of the plant, such as in the leaves, to reduce or suppress.
  • parts of a coding region used here denotes nucleotide sequences with at least 20 nucleotides which, in the antisense orientation, enable repression of an amino acid transporter gene.
  • an RNA sequence corresponding to the DNA sequence denotes an RNA sequence which has the same sequence of the purine and pyrimidine bases as a DNA sequence, but has the base uracil instead of the base thymine in the DNA sequence ,
  • a mixed sequence composed of DNA and RNA nucleotides corresponding to the DNA sequence denotes a nucleotide sequence which has the same sequence of the purine and pyrimidine bases as a DNA sequence, but which has both DNA nucleotides and RNA - Contains nucleotides, and wherein the RNA nucleotides have the base uracil instead of the base thymine in the DNA sequence.
  • Chimeric oligonucleotides represent such mixed sequences, for example.
  • Antisense orientation of a DNA sequence here means that transcription of the DNA sequence results in an mRNA whose nucleotide sequence is complementary to the natural one
  • RNA sequence is complementary to an endogenous mRNA and its translation is hindered or prevented by attachment.
  • a DNA sequence used in the method according to the invention preferably comprises the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence complementary to this nucleotide sequence. sequence, or hybridizes with the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence that is complementary to the nucleotide sequence of SEQ ID NO: 5.
  • the DNA sequence can also, alternatively or additionally, the nucleotide sequence (s) of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO : 7 or SEQ ID NO: 8 or a nucleotide sequence complementary to this nucleotide sequence, or with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a nucleotide sequence which corresponds to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 is complementary, hybridize.
  • hybridizing means hybridizing under usual conditions, as described in Sa brook et al. (Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd ed., 1989), preferably under stringent conditions.
  • Stringent hybridization conditions are, for example: hybridization in 4 x SSC at 65 ° C and subsequent multiple washing in 0.1 x SSC at 65 ° C for a total of about 1 hour.
  • Hybridization conditions that are not very stringent are, for example: hybridization in 4 x SSC at 37 ° C. and then repeated washing in 1 x SSC at room temperature.
  • stringent hybridization conditions used here can also mean: hybridization at 68 ° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequent washing twice with 2 ⁇ SSC and 0.1% SDS at 68 ° C.
  • the invention also relates to DNA sequences which contain the coding region for an amino acid transporter or parts thereof, RNA sequences corresponding to the DNA sequences and mixed sequences composed of RNA and DNA nucleotides corresponding to the DNA sequences.
  • the DNA sequence comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, or hybridizes with the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1.
  • the DNA sequence comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or one complementary to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 Nucleotide sequence, or hybridizes with the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 ,
  • the invention also relates to vectors or mobile genetic elements which comprise at least one DNA sequence and / or at least one RNA sequence corresponding to the DNA sequence and / or at least one mixed sequence composed of DNA and RNA nucleotides according to the DNA sequence present invention included.
  • Vectors or mobile genetic elements which are suitable for introducing nucleotide sequences into host cells, for example viruses, bacteriophages, cosmids, plasmids, artificial yeast chromosomes, T-DNA, transposons, insertion sequences etc., are well known to those skilled in the field of molecular cloning techniques.
  • the DNA sequence (s) according to the invention and / or the RNA sequence (s) corresponding to the DNA sequence (s) and / or the DNA sequence (s) is / are preferred ) contain corresponding mixed sequence (s) of DNA and RNA nucleotides in antisense orientation in the vector or the mobile genetic element.
  • the RNA sequence (s) are / are contained in an RNA double strand in the vector or the mobile genetic element. This enables the endogenous amino acid transporter gene to be muted by RNA interference.
  • the invention further relates to eukaryotic or prokaryotic host cells which contain at least one DNA and / or at least one RNA sequence corresponding to this and / or at least one mixed sequence composed of DNA and RNA nucleotides corresponding to the DNA sequence according to the present invention , wherein the DNA, RNA and / or DNA-RNA mixed sequence (s) is / are preferably contained in the host cell in an antisense orientation.
  • RNA sequence (s) are / are contained in an RNA double strand in the eukaryotic or prokaryotic host cell.
  • the invention also relates to plants and parts or seeds of plants which are composed of at least one DNA sequence and / or at least one RNA sequence corresponding to the DNA sequence and / or at least one corresponding to the DNA sequence of DNA and RNA nucleotides Mixing sequence are transformed according to the present invention.
  • the plants can, for example, be plants of the Beta genus, preferably of the Beta vulgaris species.
  • numerous other plants are also possible, for example potatoes, tomatoes, sugar cane, tobacco, rapeseed, ricinus, etc.
  • plants are available in which the expression of an amino acid transporter gene, for example by means of co-suppression through interaction with a homologous ectopic (out of position) gene sequence is suppressed.
  • the transgenic plants, their parts or their seeds are preferably transformed with the DNA and / or RNA and / or DNA-RNA mixing sequence in the antisense orientation.
  • the transcription of the DNA sequence in the antisense orientation forms an mRNA which at least partially hybridizes with the naturally formed mRNA for the amino acid transporter in such a way that translation into the corresponding protein cannot take place.
  • the introduction of an RNA sequence in an antisense orientation leads to an attachment of the RNA sequence to the naturally formed mRNA, so that the translation of the mRNA is also impeded in this case.
  • transgenic plants their parts or their seeds, which have been transformed with an RNA sequence in an RNA double strand.
  • the present invention further relates to the use of DNA, RNA and / or DNA-RNA mixed sequences according to the invention for producing a transgenic plant cell or plant with an increased expression of the coding region of the amino acid transporter compared to the wild type.
  • the sequences according to the invention are used in a sense orientation. This makes it possible, for example, to obtain plants which, compared to wild-type plants, increasingly incorporate nitrogen compounds. In conjunction with suitable promoters, it is also possible to influence the increased incorporation of nitrogen compounds in a tissue-specific manner. In this way, plants, for example soybean plants, can be produced whose harvestable parts have an increased content of organic nitrogen compounds compared to wild type plants.
  • the invention further relates to the use of the DNA sequence and / or a corresponding RNA sequence and / or a mixed sequence corresponding to the DNA sequence and composed of DNA and RNA nucleotides according to the present invention in an antisense orientation for the production of a transgenic plant cell or Plant with a reduced expression of the coding region of the amino acid transporter compared to the wild type.
  • transgenic plants have a reduced ability compared to wild type plants to store organic nitrogen compounds in storage organs.
  • the organic nitrogen compounds predominantly formed in the leaves are not or only to a lesser extent transported into the storage organs in the senescence phase, since the required amino acid transporter molecules are not available in sufficient quantities.
  • sequence listing (according to WIPO standard St. 25) contains:
  • SEQ ID NO: 1 A nucleotide sequence of the coding region of the AAPI gene from Beta vulgaris.
  • SEQ ID NO: 2 A nucleotide sequence of the coding region of the AAP6 gene from Beta vulgaris.
  • SEQ ID NO: 3 A nucleotide sequence of the coding region of the AAP2 gene from Beta vulgaris.
  • SEQ ID NO: 4 A nucleotide sequence of the coding region of the AAP3 gene from Beta vulgaris.
  • SEQ ID NO: 5 A nucleotide sequence of the coding region of the GAPI gene from Beta vulgaris.
  • SEQ ID NO: 6 A nucleotide sequence of the coding region of another AAP gene from Beta vulgaris.
  • SEQ ID NO: 7 A nucleotide sequence (BvProT-like) of the coding region of a further AAP gene from Beta vulgaris.
  • SEQ ID NO: 8 A nucleotide sequence (BvSV2-like) of the coding region of another AAP gene from Beta vulgaris.
  • BvGAPl The amino acid transporter encoded by SEQ ID NO: 5 (BvGAPl) has proven to be a transporter with a wide range of amino acids. "GAP” therefore also stands for "general amino acid permease”. BvGAPl transports gamma-aminobutyric acid (GABA) particularly effectively. Acidic amino acids such as aspartate are also transported particularly well, citrulline, lysine and histidine are also transported,
  • amino acid transporters encoded by SEQ ID NO: 7 (BvProT-like) and SEQ ID NO: 8 (BvSV2-like) specifically transport proline, citrulline and gamma-aminobutyric acid (GABA).
  • FIG. 1 shows: Expression of BvAAP genes (BvAAPl, BvAAP2 and BvAAP6) in different senescence stages of leaves and in five and six month old storage roots of Beta vulgaris, determined by Northern hybridization. 18s rRNA served as a control. It means: leaf RNA: RNA from leaves beet RNA: RNA from storage roots
  • RT-PCR reverse transcription followed by polymerase chain reaction
  • three nucleotide sequences were isolated and sequenced from senescent leaves and storage root material of the sugar beet (beta vulgaris), which encode the coding region of amino acid transporters (BvAAPl and BvAAP2, BvAAP6), or parts thereof , include.
  • CDNA obtained from the nucleotide sequences was cloned into the yeast expression vector pDRl96.
  • the vector pBin19 and derivatives thereof are suitable as vectors for the transformation of sugar beets. Again, cloning in antisense Orientation possible, for example, under the control of the CaMV-35S Pro otor.

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Abstract

The invention relates to a method for producing a transgenic plant, whereby the capacity for storing undesired nitrogen compounds, especially amino acids, in the storage organs is reduced in comparison with the wild type. Said method consists of the following steps; introduction of at least one DNA-sequence and/or at least one RNA-sequence corresponding to the DNA-sequence and/or one mixed sequence made of DNA and RNA nucleotide corresponding to the DNA-sequence with a coding region for an amino acid transporter or parts thereof into a plant cell. The DNA and/or RNA sequence and/or mixed sequence is used in a sense or antisense orientation and the expression of an endogenic amino acid transporter gene is prevented or reduced. The method then regenerates a plant from said plant cells, whereby the DNA and/or RNA sequence is a sequence from Beta vulgaris.

Description

VERFAHREN ZUR HERSTELLUNG EINER TRANSGENEN PFLANZE MIT VERÄNDERTEM STOFFTRANSPORTMETHOD FOR PRODUCING A TRANSGENIC PLANT WITH A CHANGED SUBSTANCE
Verfahren zur Herstellung einer transgenen Pflanze mit verändertem StofftransportProcess for the production of a transgenic plant with an altered mass transport
Die Erfindung betrifft ein Verfahren zur Herstellung einer transgenen Pflanze, bei der die Fähigkeit zur Einlagerung unerwünschter StickstoffVerbindungen, insbesondere Aminosäuren, in den Ernte- und/oder Vermehrungsorganen gegenüber dem Wildtyp vermindert ist. Die Erfindung betrifft weiterhin DNA-Sequenzen sowie diesen entsprechende RNA-Sequenzen, die die codierende Region für einen Aminosäuretransporter oder Teile davon enthalten, deren Verwendung sowie Vektoren, bewegliche genetische Elemente, Bakterien, Wirtszellen, Pflanzenzellen, Pflanzen, Samen und anderes Vermehrungsmaterial von Pflanzen, die die DNA- und/oder RNA-Sequenzen enthalten.The invention relates to a method for producing a transgenic plant in which the ability to store undesired nitrogen compounds, in particular amino acids, in the harvesting and / or reproductive organs is reduced compared to the wild type. The invention further relates to DNA sequences and RNA sequences corresponding to them, which contain the coding region for an amino acid transporter or parts thereof, their use and vectors, mobile genetic elements, bacteria, host cells, plant cells, plants, seeds and other plant propagation material, which contain the DNA and / or RNA sequences.
Pflanzen nehmen Stickstoff, der zum Aufbau von Zellsubstanz benötigt wird, vorwiegend in Form von Nitrat, aber auch als Ammonium und, in geringem Maße, als Aminosäuren, beispielsweise über die Wurzeln auf. Das Nitrat wird zu Aminostick- stoff reduziert und in organische Verbindungen eingebaut. Dies geschieht überwiegend in den Blättern der Pflanze. Stickstoffhaltige organische Verbindungen, beispielsweise Aminosäuren, werden über ein Gefäßsystem, das Phloe , von Bildungsorten wie den Blättern zu Verbrauchsgeweben und -Organen, beispielsweise Vermehrungsorganen oder Speicherorganen (Wurzeln, Knollen, Rüben usw.), transportiert.Plants absorb nitrogen, which is required to build up cell substance, mainly in the form of nitrate, but also as ammonium and, to a lesser extent, as amino acids, for example via the roots. The nitrate is reduced to amino nitrogen and incorporated into organic compounds. This mainly happens in the leaves of the plant. Nitrogen-containing organic compounds, for example amino acids, are transported via a vascular system, the phloe, from places of formation such as the leaves to consumption tissues and organs, for example reproductive organs or storage organs (roots, tubers, beets, etc.).
Die Transportprozesse sind insbesondere auch bedeutsam bei der Verlagerung von Stickstoffverbindungen im Zuge der Alterung (Seneszenz) von Blättern, etwa gegen Ende der Vegetationsperiode. Dabei werden Stickstoffverbindungen aus den absterbenden Blättern in andere Organe, beispielsweise Speieherorgane, verlagert, um so den Verlust an Zellmaterial möglichst gering zu halten.The transport processes are particularly important for the shifting of nitrogen compounds in the course of the aging (senescence) of leaves, for example towards the end of the growing season. This removes nitrogen compounds from the dying leaves into other organs, for example Storage organs, relocated to minimize the loss of cell material.
Der Transport von Aminosäuren wird von Proteinen vermittelt, sogenannten Aminosäuretransportern, zu denen die Aminosäure- permeasen (AAPs = amino acid permeases) gehören. AAPs vermitteln nicht nur den Transport eines breiten Spektrums von Aminosäuren mit vergleichsweise geringer Selektivität, sondern darüber hinaus auch von anderen organischen Stickstoffverbindungen, beispielsweise Amide wie Glutamin und Asparagin sowie von Citrullin, Gamma-Aminobuttersäure oder Auxin. Ihnen kommt daher vermutlich eine Schlüsselrolle bei der Verteilung organischer Stickstoffverbindungen innerhalb der Pflanze zu.The transport of amino acids is mediated by proteins, so-called amino acid transporters, to which the amino acid permeases (AAPs = amino acid permeases) belong. AAPs not only mediate the transport of a broad spectrum of amino acids with comparatively low selectivity, but also of other organic nitrogen compounds, for example amides such as glutamine and asparagine as well as citrulline, gamma-aminobutyric acid or auxin. They therefore presumably play a key role in the distribution of organic nitrogen compounds within the plant.
Aminosäuretransportergene aus Arabidopsis thaliana sind beispielsweise aus EP 0652955 bekannt. AAP-Gene aus der Zuckerrübe {Beta vulgaris) sind dagegen noch nicht isoliert worden.Amino acid transporter genes from Arabidopsis thaliana are known for example from EP 0652955. AAP genes from the sugar beet (beta vulgaris), however, have not yet been isolated.
Die Einlagerung von organischen Stickstoffverbindungen wie Proteinen und Aminosäuren in Ernteorganen von Pflanzen ist vielfach unerwünscht. Aufgabe der vorliegenden Erfindung ist es daher, eine Möglichkeit zu schaffen, Pflanzen herzustellen, bei denen die Einlagerung organischer Stickstoffverbindungen in ihren Ernteorganen gegenüber dem Wildtyp vermindert ist.The storage of organic nitrogen compounds such as proteins and amino acids in crop organs of plants is often undesirable. The object of the present invention is therefore to create a possibility of producing plants in which the incorporation of organic nitrogen compounds in their harvesting organs is reduced compared to the wild type.
Diese Aufgabe wird gelöst durch ein Verfahren, das die Schritte Einführen mindestens einer DNA-Sequenz und/oder einer der DNA-Sequenz entsprechenden RNA-Sequenz und/oder einer der DNA-Sequenz entsprechenden aus DNA- und RNA- Nukleotiden zusammengesetzten Mischsequenz mit der codierenden Region für einen Aminosäuretransporter oder Teilen davon in eine Pflanzenzelle, wobei die DNA- und/oder RNA-Sequenz und/oder Mischsequenz in Sense- oder Antisense-Orientierung verwendet und die Expression eines endogenen Aminosauretransportergens verhindert oder reduziert wird, und Regenerieren einer Pflanze aus dieser Pflanzenzelle umfaßt, wobei die DNA- und/oder RNA-Sequenz eine Sequenz aus Beta vulgaris ist.This object is achieved by a method which comprises the steps of introducing at least one DNA sequence and / or an RNA sequence corresponding to the DNA sequence and / or a mixed sequence composed of DNA and RNA nucleotides with the coding sequence Region for an amino acid transporter or parts thereof in a plant cell, the DNA and / or RNA sequence and / or mixed sequence in sense or antisense orientation is used and the expression of an endogenous amino acid transporter gene is prevented or reduced, and regenerating a plant from this plant cell, wherein the DNA and / or RNA sequence is a sequence from Beta vulgaris.
Pflanzen, die nach dem erfindungsgemäßen Verfahren hergestellt wurden, weisen einen deutlich verminderten Gehalt an organischen Stickstoffverbindungen, insbesondere Aminosäuren, in ihren Ernteorganen auf. In einer Pflanzenzelle, in die die DNA-Sequenz und/oder die der DNA-Sequenz entsprechende RNA- Sequenz und/oder die der DNA-Sequenz entsprechende RNA-/DNA- Mischsequenz in Antisense-Orientierung eingeführt wurde, ist die Translation der mRNA des endogenen Aminosauretransportergens durch Anlagerung einer Antisense-RNA an die RNA behindert. Im Falle der Einführung einer DNA-Sequenz wird die Antisense-RNA durch Transkription der DNA-Sequenz gebildet. Im Falle der der DNA-Sequenz entsprechenden RNA-Sequenz kann diese selbst die Antisense-RNA darstellen. Eine posttrans- kriptionelle Stummschaltung (PTGS = posttranscriptional gene silencing) des endogenen Aminosauretransportergens kann auch durch Co-Suppression oder RNA-Interferenz herbeigeführt werden, indem die DNA-Sequenz oder eine der DNA-Sequenz entsprechende RNA-Sequenz in Sense-Orientierung in die Pflanzenzelle eingeführt wird, wobei die RNA im Falle der RNA-Interferenz als Doppelstrang-RNA verwendet wird. Darüber hinaus ist es auch möglich, aus RNA- und DNA-Sequenzen zusammengesetzte Mischsequenzen, chimäre Oligonukleotide, wie sie beispielsweise von Rice et al . (2000), Plant Physiology 123, 427-437, beschrieben werden, einzusetzen, um ein Stummschalten des endogenen Aminosauretransportergens zu bewirken. Die nach dem erfindungsgemäßen Verfahren hergestellten Pflanzen sind auf diese Weise weitgehend daran gehindert, Aminosäuretransporter zu bilden. Durch eine Kopplung an geeignete gewebespezifische Promotoren ist es auch möglich, gezielt die Bildung von Aminosäuretransportern in bestimmten Geweben oder Organen der Pflanze, etwa in den Blättern, zu vermindern oder zu unterdrücken.Plants which have been produced by the process according to the invention have a significantly reduced content of organic nitrogen compounds, in particular amino acids, in their harvesting organs. In a plant cell into which the DNA sequence and / or the RNA sequence corresponding to the DNA sequence and / or the RNA / DNA mixed sequence corresponding to the DNA sequence has been introduced in the antisense orientation, the translation of the mRNA of the endogenous amino acid transporter gene hindered by the attachment of an antisense RNA to the RNA. If a DNA sequence is introduced, the antisense RNA is formed by transcription of the DNA sequence. In the case of the RNA sequence corresponding to the DNA sequence, this can itself represent the antisense RNA. Post-transcriptional muting (PTGS = post-transcriptional gene silencing) of the endogenous amino transporter gene can also be brought about by co-suppression or RNA interference, in that the DNA sequence or an RNA sequence corresponding to the DNA sequence is sense-oriented into the plant cell is introduced, the RNA being used as double-stranded RNA in the case of RNA interference. In addition, it is also possible to use mixed sequences composed of RNA and DNA sequences, chimeric oligonucleotides, as described, for example, by Rice et al. (2000), Plant Physiology 123, 427-437, can be used to mute the endogenous amino transporter gene. The plants produced by the process according to the invention are largely prevented in this way from forming amino acid transporters. By coupling to suitable tissue-specific promoters, it is also possible to specifically target the formation of amino acid transporters in certain tissues or organs of the plant, such as in the leaves, to reduce or suppress.
Der hier verwendete Begriff "Teile" einer codierenden Region bezeichnet Nukleotidsequenzen mit mindestens 20 Nukleotiden, die in Antisense-Orientierung eine Repression eines Aminosauretransportergens möglich machen.The term “parts” of a coding region used here denotes nucleotide sequences with at least 20 nucleotides which, in the antisense orientation, enable repression of an amino acid transporter gene.
Der hier verwendete Begriff "eine der DNA-Sequenz entsprechende RNA-Sequenz" bezeichnet eine RNA-Sequenz, die die gleiche Abfolge der Purin- und Pyrimidinbasen wie eine DNA- Sequenz, anstelle der Base Thymin in der DNA-Sequenz aber die Base Uracil aufweist.The term “an RNA sequence corresponding to the DNA sequence” used here denotes an RNA sequence which has the same sequence of the purine and pyrimidine bases as a DNA sequence, but has the base uracil instead of the base thymine in the DNA sequence ,
Der hier verwendete Begriff "eine der DNA-Sequenz entsprechende aus DNA- und RNA-Nukleotiden zusammengesetzte Mischsequenz" bezeichnet eine Nukleotidsequenz, die die gleiche Abfolge der Purin- und Pyrimidinbasen wie eine DNA-Sequenz aufweist, die aber sowohl DNA-Nukleotide als auch RNA- Nukleotide enthält, und wobei die RNA-Nukleotide statt der Base Thymin in der DNA-Sequenz die Base Uracil aufweisen. Chimäre Oligonukleotide stellen beispielsweise solche Mischsequenzen dar.The term "a mixed sequence composed of DNA and RNA nucleotides corresponding to the DNA sequence" used here denotes a nucleotide sequence which has the same sequence of the purine and pyrimidine bases as a DNA sequence, but which has both DNA nucleotides and RNA - Contains nucleotides, and wherein the RNA nucleotides have the base uracil instead of the base thymine in the DNA sequence. Chimeric oligonucleotides represent such mixed sequences, for example.
"Antisense-Orientierung" einer DNA-Sequenz bedeutet hier, daß eine Transkription der DNA-Sequenz in einer mRNA resultiert, deren Nukleotidsequenz komplementär ist zu der natürlichen"Antisense orientation" of a DNA sequence here means that transcription of the DNA sequence results in an mRNA whose nucleotide sequence is complementary to the natural one
(endogenen) mRNA, so daß deren Translation behindert oder verhindert wird. "Antisense-Orientierung" einer RNA-Sequenz bedeutet, daß die RNA-Sequenz zu einer endogenen mRNA komplementär ist und deren Translation durch Anlagerung behindert oder verhindert .(endogenous) mRNA, so that their translation is hindered or prevented. "Antisense orientation" of an RNA sequence means that the RNA sequence is complementary to an endogenous mRNA and its translation is hindered or prevented by attachment.
Bevorzugt umfaßt eine in dem erfindungsgemäßen Verfahren eingesetzte DNA-Sequenz die Nukleotidsequenz der SEQ ID NO: 5 oder eine zu dieser Nukleotidsequenz komplementäre Nukleotid- sequenz, oder hybridisiert mit der Nukleotidsequenz der SEQ ID NO: 5 oder einer Nukleotidsequenz, die zu der Nukleotidsequenz der SEQ ID NO: 5 komplementär ist. Die DNA-Sequenz kann auch, alternativ oder zusätzlich, die Nukleotidsequenz (en) der SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO: 4, SEQ ID NO : 6, SEQ ID NO : 7 bzw. SEQ ID NO : 8 oder eine zu dieser Nukleotidsequenz komplementäre Nukleotidsequenz umfassen, oder mit der Nukleotidsequenz der SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO: 6, SEQ ID NO: 7 bzw. SEQ ID NO : 8 oder einer Nukleotidsequenz, die zu der Nukleotidsequenz der SEQ ID NO : 1, SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 7 bzw. SEQ ID NO: 8 komplementär ist, hybridisieren.A DNA sequence used in the method according to the invention preferably comprises the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence complementary to this nucleotide sequence. sequence, or hybridizes with the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence that is complementary to the nucleotide sequence of SEQ ID NO: 5. The DNA sequence can also, alternatively or additionally, the nucleotide sequence (s) of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO : 7 or SEQ ID NO: 8 or a nucleotide sequence complementary to this nucleotide sequence, or with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a nucleotide sequence which corresponds to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 is complementary, hybridize.
Der hier verwendete Begriff "Hybridisieren" bedeutet Hybridisieren unter üblichen Bedingungen, wie sie in Sa brook et al . (Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, 2. Aufl., 1989) beschrieben sind, bevorzugt unter stringenten Bedingungen. Stringente Hybridisierungsbe- dingungen sind beispielsweise: Hybridisieren in 4 x SSC bei 65 °C und anschließendes mehrfaches Waschen in 0,1 x SSC bei 65 °C für insgesamt etwa 1 Stunde. Wenig stringente Hybridi- sierungsbedingungen sind beispielsweise: Hybridisieren in 4 x SSC bei 37 °C und anschließendes mehrfaches Waschen in 1 x SSC bei Raumtemperatur. Der hier verwendete Begriff "stringente Hybridisierungsbedingungen" kann auch bedeuten: Hybridisieren bei 68 °C in 0,25 M Natriumphosphat, pH 7,2, 7 % SDS, 1 mM EDTA und 1 % BSA für 16 Stunden und anschließendes zweimaliges Waschen mit 2 x SSC und 0,1 % SDS bei 68 °C.The term "hybridizing" as used herein means hybridizing under usual conditions, as described in Sa brook et al. (Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd ed., 1989), preferably under stringent conditions. Stringent hybridization conditions are, for example: hybridization in 4 x SSC at 65 ° C and subsequent multiple washing in 0.1 x SSC at 65 ° C for a total of about 1 hour. Hybridization conditions that are not very stringent are, for example: hybridization in 4 x SSC at 37 ° C. and then repeated washing in 1 x SSC at room temperature. The term "stringent hybridization conditions" used here can also mean: hybridization at 68 ° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequent washing twice with 2 × SSC and 0.1% SDS at 68 ° C.
Die Erfindung betrifft auch DNA-Sequenzen, die die codierende Region für einen Aminosäuretransporter oder Teile davon enthalten, den DNA-Sequenzen entsprechende RNA-Sequenzen sowie den DNA-Sequenzen entsprechende aus RNA- und DNA- Nukleotiden zusammengesetzte Mischsequenzen. Hierbei umfaßt die DNA-Sequenz die Nukleotidsequenz der SEQ ID NO: 1 oder eine zu der Nukleotidsequenz der SEQ ID NO : 1 komplementäre Nukleotidsequenz, oder hybridisiert mit der Nukleotidsequenz der SEQ ID NO : 1 oder einer zu der Nukleotidsequenz der SEQ ID NO : 1 komplementären Nukleotidsequenz . Alternativ umfaßt die DNA-Sequenz die Nukleotidsequenz der SEQ ID NO : 2, SEQ ID NO: 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7 bzw. SEQ ID NO: 8 oder eine zu der Nukleotidsequenz der SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO : 7 bzw. SEQ ID NO : 8 komplementäre Nukleotidsequenz, oder hybridisiert mit der Nukleotidsequenz der SEQ ID NO : 2, SEQ ID NO: 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO : 7 bzw. SEQ ID NO : 8 oder einer zu der Nukleotidsequenz der SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO: 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7 bzw. SEQ ID NO: 8 komplementären Nukleotidsequenz.The invention also relates to DNA sequences which contain the coding region for an amino acid transporter or parts thereof, RNA sequences corresponding to the DNA sequences and mixed sequences composed of RNA and DNA nucleotides corresponding to the DNA sequences. Here, the DNA sequence comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, or hybridizes with the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1. Alternatively, the DNA sequence comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or one complementary to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 Nucleotide sequence, or hybridizes with the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 ,
Die Erfindung betrifft auch Vektoren oder mobile genetische Elemente, die mindestens eine DNA-Sequenz und/oder mindestens eine der DNA-Sequenz entsprechende RNA-Sequenz und/oder mindestens eine der DNA-Sequenz entsprechende aus DNA- und RNA-Nukleotiden zusammengesetzte Mischsequenz gemäß der vorliegenden Erfindung enthalten. Vektoren oder mobile genetische Elemente, die zur Einführung von Nukleotid- sequenzen in Wirtszellen geeignet sind, beispielsweise Viren, Bakteriophagen, Cosmide, Plasmide, künstliche Hefechromosomen, T-DNA, Transposons, Insertionssequenzen usw. , sind dem Fachmann auf dem Gebiet molekularer Klonierungstechniken wohlbekannt .The invention also relates to vectors or mobile genetic elements which comprise at least one DNA sequence and / or at least one RNA sequence corresponding to the DNA sequence and / or at least one mixed sequence composed of DNA and RNA nucleotides according to the DNA sequence present invention included. Vectors or mobile genetic elements which are suitable for introducing nucleotide sequences into host cells, for example viruses, bacteriophages, cosmids, plasmids, artificial yeast chromosomes, T-DNA, transposons, insertion sequences etc., are well known to those skilled in the field of molecular cloning techniques.
Bevorzugt ist/sind die erfindungsgemäße (n) DNA-Sequenz (en) und/oder die der/den DNA-Sequenz (en) entsprechende (n) RNA- Sequenz (en) und/oder die der/den DNA-Sequenz (en) entsprechende (n) aus DNA- und RNA-Nukleotiden zusammengesetzte (n) Mischsequenz (en) in Antisense-Orientierung in dem Vektor oder dem mobilen genetischen Element enthalten. In einer weiteren bevorzugten Ausführungsform ist/sind die RNA-Sequenz (en) in einem RNA-Doppelstrang in dem Vektor oder dem mobilen genetischen Element enthalten. Dies ermöglicht die Stummschaltung des endogenen Aminosauretransportergens durch RNA-Interferenz .The DNA sequence (s) according to the invention and / or the RNA sequence (s) corresponding to the DNA sequence (s) and / or the DNA sequence (s) is / are preferred ) contain corresponding mixed sequence (s) of DNA and RNA nucleotides in antisense orientation in the vector or the mobile genetic element. In a further preferred embodiment, the RNA sequence (s) are / are contained in an RNA double strand in the vector or the mobile genetic element. This enables the endogenous amino acid transporter gene to be muted by RNA interference.
Ferner betrifft die Erfindung eukaryotische oder prokaryo- tische Wirtszellen, die mindestens eine DNA- und/oder mindestens eine dieser entsprechenden RNA-Sequenz und/oder mindestens eine der DNA-Sequenz entsprechende aus DNA- und RNA-Nukleotiden zusammengesetzte Mischsequenz gemäß der vorliegenden Erfindung enthalten, wobei die DNA-, RNA- und/oder DNA-RNA-Mischsequenz (en) bevorzugt in Antisense- Orientierung in der Wirtszelle enthalten ist/sind.The invention further relates to eukaryotic or prokaryotic host cells which contain at least one DNA and / or at least one RNA sequence corresponding to this and / or at least one mixed sequence composed of DNA and RNA nucleotides corresponding to the DNA sequence according to the present invention , wherein the DNA, RNA and / or DNA-RNA mixed sequence (s) is / are preferably contained in the host cell in an antisense orientation.
In einer weiteren bevorzugten Ausführungsform ist/sind die RNA-Sequenz (en) in einem RNA-Doppelstrang in der eukaryo- tischen oder prokaryotischen Wirtszelle enthalten.In a further preferred embodiment, the RNA sequence (s) are / are contained in an RNA double strand in the eukaryotic or prokaryotic host cell.
Die Erfindung betrifft auch Pflanzen sowie Teile oder Samen von Pflanzen, die mit mindestens einer DNA-Sequenz und/oder mindestens einer der DNA-Sequenz entsprechenden RNA-Sequenz und/oder mindestens einer der DNA-Sequenz entsprechenden aus DNA- und RNA-Nukleotiden zusammengesetzten Mischsequenz gemäß der vorliegenden Erfindung transformiert sind.The invention also relates to plants and parts or seeds of plants which are composed of at least one DNA sequence and / or at least one RNA sequence corresponding to the DNA sequence and / or at least one corresponding to the DNA sequence of DNA and RNA nucleotides Mixing sequence are transformed according to the present invention.
Die Pflanzen können beispielsweise Pflanzen der Gattung Beta, bevorzugt der Art Beta vulgaris, sein. Es kommen aber auch zahlreiche andere Pflanzen in Frage, beispielsweise Kartoffeln, Tomaten, Zuckerrohr, Tabak, Raps, Ricinus usw. Durch Transformieren der Pflanzen, Pflanzenteile oder Pflanzensamen mit einer erfindungsgemäßen DNA-Sequenz, RNA- Sequenz oder DNA-RNA-Mischsequenz sind Pflanzen erhältlich, bei denen die Expression eines Aminosauretransportergens beispielsweise mittels Co-Suppression durch Interaktion mit einer homologen ektopischen (nicht an der normalen Position gelegenen) Gensequenz unterdrückt wird.The plants can, for example, be plants of the Beta genus, preferably of the Beta vulgaris species. However, numerous other plants are also possible, for example potatoes, tomatoes, sugar cane, tobacco, rapeseed, ricinus, etc. By transforming the plants, parts of plants or plant seeds with a DNA sequence, RNA sequence or DNA-RNA mixed sequence according to the invention, plants are available in which the expression of an amino acid transporter gene, for example by means of co-suppression through interaction with a homologous ectopic (out of position) gene sequence is suppressed.
Bevorzugt sind die transgenen Pflanzen, deren Teile oder deren Samen mit der DNA- und/oder RNA- und/oder DNA-RNA- Mischsequenz in Antisense-Orientierung transformiert. Durch die Transkription der DNA-Sequenz in Antisense-Orientierung wird eine mRNA gebildet, die mit der natürlicherweise gebildeten mRNA für den Aminosäuretransporter zumindest teilweise so hybridisiert, daß eine Translation in das entsprechende Protein nicht erfolgen kann. Das Einführen einer RNA-Sequenz in Antisense-Orientierung führt zu einer Anlagerung der RNA- Sequenz an die natürlicherweise gebildete mRNA, so daß auch in diesem Fall die Translation der mRNA behindert wird.The transgenic plants, their parts or their seeds are preferably transformed with the DNA and / or RNA and / or DNA-RNA mixing sequence in the antisense orientation. The transcription of the DNA sequence in the antisense orientation forms an mRNA which at least partially hybridizes with the naturally formed mRNA for the amino acid transporter in such a way that translation into the corresponding protein cannot take place. The introduction of an RNA sequence in an antisense orientation leads to an attachment of the RNA sequence to the naturally formed mRNA, so that the translation of the mRNA is also impeded in this case.
Weiter bevorzugt sind transgene Pflanzen, deren Teile oder deren Samen, die mit einer RNA-Sequenz in einem RNA-Doppelstrang transformiert wurden.Further preferred are transgenic plants, their parts or their seeds, which have been transformed with an RNA sequence in an RNA double strand.
Die vorliegende Erfindung betrifft weiterhin die Verwendung von erfindungsgemäßen DNA-, RNA- und/oder DNA-RNA-Mischse- quenzen zur Herstellung einer transgenen Pflanzenzelle oder Pflanze mit einer gegenüber dem Wildtyp erhöhten Expression der codierenden Region des Aminosäuretransporters. Die erfindungsgemäßen Sequenzen werden dabei in Sense- Orientierung verwendet. Hierdurch ist es beispielsweise möglich, Pflanzen zu erhalten, die gegenüber Pflanzen des Wildtyps vermehrt Stickstoffverbindungen einlagern. In Verbindung mit geeigneten Promotoren ist es auch möglich, die vermehrte Einlagerung von Stickstoffverbindungen gewebespezifisch zu beeinflussen. Auf diese Weise sind Pflanzen, beispielsweise Sojapflanzen, herstellbar, deren erntbare Teile einen gegenüber Wildtyppflanzen erhöhten Gehalt an organischen Stickstoffverbindungen aufweisen. Weiter betrifft die Erfindung die Verwendung der DNA-Sequenz und/oder einer entsprechenden RNA-Sequenz und/oder einer der DNA-Sequenz entsprechenden aus DNA- und RNA-Nukleotiden zusammengesetzten Mischsequenz gemäß der vorliegenden Erfindung in Antisense-Orientierung zur Herstellung einer transgenen Pflanzenzelle oder Pflanze mit einer gegenüber dem Wildtyp verminderten Expression der codierenden Region des Aminosäuretransporters. Solche transgenen Pflanzen weisen gegenüber Wildtyppflanzen eine verminderte Fähigkeit auf, organische Stickstoffverbindungen in Speicherorgane einzulagern. Die vorwiegend in den Blättern gebildeten organischen Stickstoffverbindungen werden auch in der Phase der Seneszenz nicht oder nur in geringerem Umfang in die Speicherorgane transportiert, da die erforderlichen Aminosäuretransporter-Moleküle nicht in ausreichender Menge zur Verfügung stehen.The present invention further relates to the use of DNA, RNA and / or DNA-RNA mixed sequences according to the invention for producing a transgenic plant cell or plant with an increased expression of the coding region of the amino acid transporter compared to the wild type. The sequences according to the invention are used in a sense orientation. This makes it possible, for example, to obtain plants which, compared to wild-type plants, increasingly incorporate nitrogen compounds. In conjunction with suitable promoters, it is also possible to influence the increased incorporation of nitrogen compounds in a tissue-specific manner. In this way, plants, for example soybean plants, can be produced whose harvestable parts have an increased content of organic nitrogen compounds compared to wild type plants. The invention further relates to the use of the DNA sequence and / or a corresponding RNA sequence and / or a mixed sequence corresponding to the DNA sequence and composed of DNA and RNA nucleotides according to the present invention in an antisense orientation for the production of a transgenic plant cell or Plant with a reduced expression of the coding region of the amino acid transporter compared to the wild type. Such transgenic plants have a reduced ability compared to wild type plants to store organic nitrogen compounds in storage organs. The organic nitrogen compounds predominantly formed in the leaves are not or only to a lesser extent transported into the storage organs in the senescence phase, since the required amino acid transporter molecules are not available in sufficient quantities.
Überraschend wurde gefunden, daß solche Pflanzen weder in ihrem Wachstum oder ihrer Entwicklung noch in ihrem sonstigen Erscheinungsbild Beeinträchtigungen aufweisen. Darüber hinaus hat sich gezeigt, daß auch die Fähigkeit zur Einlagerung von Speicherstoffen wie z.B. Stärke oder Saccharose bei diesen Pflanzen nicht beeinträchtigt ist. Vielmehr ist deren Speicherfähigkeit gegenüber Wildtyppflanzen sogar noch erhöht .Surprisingly, it was found that such plants have no impairment either in their growth or development or in their other appearance. In addition, it has been shown that the ability to store storage materials such as e.g. Starch or sucrose is not affected in these plants. Rather, their storage capacity compared to wild-type plants is even increased.
Das Sequenzprotokoll (nach WIPO-Standard St. 25) enthält:The sequence listing (according to WIPO standard St. 25) contains:
SEQ ID NO: 1: Eine Nukleotidsequenz der codierenden Region des AAPl-Gens von Beta vulgaris .SEQ ID NO: 1: A nucleotide sequence of the coding region of the AAPI gene from Beta vulgaris.
SEQ ID NO: 2: Eine Nukleotidsequenz der codierenden Region des AAP6-Gens von Beta vulgaris .SEQ ID NO: 2: A nucleotide sequence of the coding region of the AAP6 gene from Beta vulgaris.
SEQ ID NO: 3: Eine Nukleotidsequenz der codierenden Region des AAP2-Gens von Beta vulgaris . SEQ ID NO: 4: Eine Nukleotidsequenz der codierenden Region des AAP3-Gens von Beta vulgaris .SEQ ID NO: 3: A nucleotide sequence of the coding region of the AAP2 gene from Beta vulgaris. SEQ ID NO: 4: A nucleotide sequence of the coding region of the AAP3 gene from Beta vulgaris.
SEQ ID NO: 5: Eine Nukleotidsequenz der codierenden Region des GAPl-Gens von Beta vulgaris .SEQ ID NO: 5: A nucleotide sequence of the coding region of the GAPI gene from Beta vulgaris.
SEQ ID NO: 6: Eine Nukleotidsequenz der codierenden Region eines weiteren AAP-Gens von Beta vulgaris .SEQ ID NO: 6: A nucleotide sequence of the coding region of another AAP gene from Beta vulgaris.
SEQ ID NO: 7: Eine Nukleotidsequenz (BvProT-like) der codierenden Region eines weiteren AAP-Gens von Beta vulgaris .SEQ ID NO: 7: A nucleotide sequence (BvProT-like) of the coding region of a further AAP gene from Beta vulgaris.
SEQ ID NO: 8: Eine Nukleotidsequenz (BvSV2-like) der codierenden Region eines weiteren AAP-Gens von Beta vulgaris .SEQ ID NO: 8: A nucleotide sequence (BvSV2-like) of the coding region of another AAP gene from Beta vulgaris.
Der durch die SEQ ID NO : 5 (BvGAPl) codierte Aminosäuretransporter hat sich als Transporter mit einem breiten Aminosäurespektrum erwiesen. "GAP" steht daher auch für "general amino acid permease" . BvGAPl transportiert Gamma-Aminobuttersäure (GABA) besonders effektiv. Besonders gut werden auch saure Aminosäuren wie Aspartat transportiert, Citrullin, Lysin und Histidin werden ebenfalls transportiert,The amino acid transporter encoded by SEQ ID NO: 5 (BvGAPl) has proven to be a transporter with a wide range of amino acids. "GAP" therefore also stands for "general amino acid permease". BvGAPl transports gamma-aminobutyric acid (GABA) particularly effectively. Acidic amino acids such as aspartate are also transported particularly well, citrulline, lysine and histidine are also transported,
Die durch die SEQ ID NO : 7 (BvProT-like) und SEQ ID NO: 8 (BvSV2-like) codierten Aminosäuretransporter transportieren spezifisch Prolin, Citrullin und Gamma-Aminobuttersäure (GABA) .The amino acid transporters encoded by SEQ ID NO: 7 (BvProT-like) and SEQ ID NO: 8 (BvSV2-like) specifically transport proline, citrulline and gamma-aminobutyric acid (GABA).
Die Erfindung wird im weiteren anhand eines Ausführungsbei- spiels näher erläutert, ist aber nicht auf dieses beschränkt, Das Ausführungsbeispiel wird anhand der Figur 1 näher beschrieben. Es zeigt: Fig. 1: Expression von BvAAP-Genen (BvAAPl, BvAAP2 und BvAAP6) in verschiedenen Seneszenzstadien von Blättern sowie in fünf und sechs Monate alten Speicherwurzeln von Beta vulgaris, ermittelt durch Northern-Hybridisierung. 18s rRNA diente als Kontrolle. Es bedeuten: leaf RNA: RNA aus Blättern beet RNA: RNA aus SpeicherwurzelnThe invention is explained in more detail below on the basis of an exemplary embodiment, but is not limited to this. The exemplary embodiment is described in more detail with reference to FIG. 1. It shows: Fig. 1: Expression of BvAAP genes (BvAAPl, BvAAP2 and BvAAP6) in different senescence stages of leaves and in five and six month old storage roots of Beta vulgaris, determined by Northern hybridization. 18s rRNA served as a control. It means: leaf RNA: RNA from leaves beet RNA: RNA from storage roots
Beispiel :For example:
Aus seneszenten Blättern und Speicherwurzelmaterial der Zuckerrübe (Beta vulgaris) wurden mit Hilfe der RT-PCR (Reverse Transkription mit anschließender Polymeraseketten- reaktion) drei Nukleotidsequenzen isoliert und sequenziert, die die codierende Region von Aminosäuretransportern (BvAAPl und BvAAP2 , BvAAP6) , oder Teile davon, umfassen. Aus den Nukleotidsequenzen erhaltene cDNA wurde in den Hefeexpressionsvektor pDRl96 kloniert.With the help of RT-PCR (reverse transcription followed by polymerase chain reaction), three nucleotide sequences were isolated and sequenced from senescent leaves and storage root material of the sugar beet (beta vulgaris), which encode the coding region of amino acid transporters (BvAAPl and BvAAP2, BvAAP6), or parts thereof , include. CDNA obtained from the nucleotide sequences was cloned into the yeast expression vector pDRl96.
Expressionsstudien auf Gesamt-RNA-Ebene ergaben, daß die Expression von BvAAP6 während der Seneszenz stark ansteigt (Fig. 1) . Die Transkriptmenge von BvAAPl steigt während der Seneszenz ebenfalls an. Die Transkriptmenge von BvAAP2 verändert sich demgegenüber kaum. Hierdurch wird deutlich, daß insbesondere die beiden Aminosäuretransporter BvAAPl und BvAAP6 eine entscheidende Rolle bei der Verlagerung von Stickstoffverbindungen aus alternden Blättern der Zuckerrübe in die Speicherwurzel spielen. Darüber hinaus zeigen die Ergebnisse, daß BvAAP2 ebenfalls an der Verteilung von Aminosäuren beteiligt ist.Expression studies at the total RNA level showed that the expression of BvAAP6 increases strongly during senescence (FIG. 1). The amount of BvAAPl transcripts also increases during senescence. In contrast, the amount of BvAAP2 transcripts hardly changes. This makes it clear that the two amino acid transporters BvAAPl and BvAAP6 in particular play a decisive role in the shift of nitrogen compounds from aging leaves of the sugar beet into the storage root. In addition, the results show that BvAAP2 is also involved in the distribution of amino acids.
Als Vektoren zur Transformation von Zuckerrüben sind beispielsweise der Vektor pBinl9 und Derivate hiervon geeignet. Auch hier ist eine Klonierung in Antisense- Orientierung beispielsweise unter Kontrolle des CaMV-35S- Pro otors möglich. For example, the vector pBin19 and derivatives thereof are suitable as vectors for the transformation of sugar beets. Again, cloning in antisense Orientation possible, for example, under the control of the CaMV-35S Pro otor.

Claims

- Ah -PATENTANSPRÜCHE - Ah - PATENT REQUIREMENTS
1. Verfahren zur Herstellung einer transgenen Pflanze, bei der die Fähigkeit zur Einlagerung unerwünschter Stickstoffverbindungen in den Ernteorganen gegenüber dem Wildtyp vermindert ist, umfassend die Schritte Einführen mindestens einer DNA-Sequenz und/oder mindestens einer der DNA-Sequenz entsprechenden RNA-Sequenz und/oder einer der DNA-Sequenz entsprechenden aus DNA- und RNA- Nukleotiden zusammengesetzten Mischsequenz mit der codierenden Region für einen Aminosäuretransporter oder Teilen davon in eine Pflanzenzelle, wobei die DNA- und/oder RNA-Sequenz und/oder Mischsequenz in Sense- oder Antisense- Orientierung verwendet und die Expression eines endogenen Aminosauretransportergens verhindert oder reduziert wird, und1. A method for producing a transgenic plant in which the ability to store undesired nitrogen compounds in the harvesting organs is reduced compared to the wild type, comprising the steps of introducing at least one DNA sequence and / or at least one RNA sequence corresponding to the DNA sequence and / or a mixed sequence corresponding to the DNA sequence and composed of DNA and RNA nucleotides with the coding region for an amino acid transporter or parts thereof into a plant cell, the DNA and / or RNA sequence and / or mixed sequence being in sense or antisense Orientation is used and the expression of an endogenous amino acid transporter gene is prevented or reduced, and
Regenerieren einer Pflanze aus dieser Pflanzenzelle, wobei die DNA- und/oder RNA-Sequenz eine Sequenz aus Beta vulgaris ist.Regeneration of a plant from this plant cell, the DNA and / or RNA sequence being a sequence from Beta vulgaris.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß die DNA-Sequenz2. The method according to claim 1, characterized in that the DNA sequence
a) die Nukleotidsequenz der SEQ ID NO: 5 oder eine zu der Nukleotidsequenz der SEQ ID NO : 5 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO : 5 oder einer zu der Nukleotidsequenz der SEQ ID NO: 5 komplementären Nukleotidsequenz hybridisiert, und/odera) comprises the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or hybridizes with the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5, and /or
b) die Nukleotidsequenz der SEQ ID NO: 1 oder eine zu der Nukleotidsequenz der SEQ ID NO: 1 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO : 1 oder einer zu der Nukleotidsequenz der SEQ ID NO: 1 komplementären Nukleotidsequenz hybridisiert, und/oder - AH -b) comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or hybridizes with the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, and /or - AH -
c) die Nukleotidsequenz der SEQ ID NO : 2 oder eine zu der Nukleotidsequenz der SEQ ID NO : 2 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO: 2 oder einer zu der Nukleotidsequenz der SEQ ID NO : 2 komplementären Nukleotidsequenz hybridisiert, und/oderc) comprises the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or hybridizes with the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, and /or
d) die Nukleotidsequenz der SEQ ID NO: 3 oder eine zu der Nukleotidsequenz der SEQ ID NO : 3 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO: 3 oder einer zu der Nukleotidsequenz der SEQ ID NO: 3 komplementären Nukleotidsequenz hybridisiert, und/oderd) comprises the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or hybridizes with the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3, and /or
e) die Nukleotidsequenz der SEQ ID NO: 4 oder eine zu der Nukleotidsequenz der SEQ ID NO : 4 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO: 4 oder einer zu der Nukleotidsequenz der SEQ ID NO: 4 komplementären Nukleotidsequenz hybridisiert, und/odere) comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4 or hybridizes with the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4, and /or
f) die Nukleotidsequenz der SEQ ID NO: 6 oder eine zu der Nukleotidsequenz der SEQ ID NO: 6 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO: 6 oder einer zu der Nukleotidsequenz der SEQ ID NO : 6 komplementären Nukleotidsequenz hybridisiert .f) comprises the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6 or hybridizes with the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6.
g) die Nukleotidsequenz der SEQ ID NO : 7 oder eine zu der Nukleotidsequenz der SEQ ID NO : 7 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO : 7 oder einer zu der Nukleotidsequenz der SEQ ID NO: 7 komplementären Nukleotidsequenz hybridisiert . - AS -g) comprises the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7 or hybridizes with the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7. - AS -
h) die Nukleotidsequenz der SEQ ID NO: 8 oder eine zu der Nukleotidsequenz der SEQ ID NO : 8 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO : 8 oder einer zu der Nukleotidsequenz der SEQ ID NO: 8 komplementären Nukleotidsequenz hybridisiert .h) comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 8 or hybridizes with the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 8.
3. DNA-Sequenz, die die codierende Region für einen3. DNA sequence that is the coding region for a
Aminosäuretransporter enthält, oder eine der DNA-Sequenz entsprechende RNA-Sequenz, oder eine der DNA-Sequenz entsprechende aus RNA- und DNA-Nukleotiden zusammengesetzte Mischsequenz, dadurch gekennzeichnet, daß die DNA-SequenzContains amino acid transporters, or an RNA sequence corresponding to the DNA sequence, or a mixed sequence composed of RNA and DNA nucleotides corresponding to the DNA sequence, characterized in that the DNA sequence
a) die Nukleotidsequenz der SEQ ID NO: 5 oder eine zu der Nukleotidsequenz der SEQ ID NO : 5 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO: 5 oder einer zu der Nukleotidsequenz der SEQ ID NO: 5 komplementären Nukleotidsequenz hybridisiert, odera) the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or hybridized with the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5, or
b) die Nukleotidsequenz der SEQ ID NO: 1 oder eine zu der Nukleotidsequenz der SEQ ID NO : 1 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO : 1 oder einer zu der Nukleotidsequenz der SEQ ID NO: 1 komplementären Nukleotidsequenz hybridisiert, oderb) comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or hybridizes with the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, or
c) die Nukleotidsequenz der SEQ ID NO : 2 oder eine zu der Nukleotidsequenz der SEQ ID NO : 2 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO : 2 oder einer zu der Nukleotidsequenz der SEQ ID NO: 2 komplementären Nukleotidsequenz hybridisiert, oderc) comprises the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or hybridizes with the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, or
d) die Nukleotidsequenz der SEQ ID NO : 3 oder eine zu der Nukleotidsequenz der SEQ ID NO : 3 komplementäre - Ab -d) the nucleotide sequence of SEQ ID NO: 3 or one complementary to the nucleotide sequence of SEQ ID NO: 3 - from -
Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO : 3 oder einer zu der Nukleotidsequenz der SEQ ID NO: 3 komplementären Nukleotidsequenz hybridisiert, oderNucleotide sequence comprises or hybridizes with the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3, or
e) die Nukleotidsequenz der SEQ ID NO: 4 oder eine zu der Nukleotidsequenz der SEQ ID NO: 4 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO: 4 oder einer zu der Nukleotidsequenz der SEQ ID NO: 4 komplementären Nukleotidsequenz hybridisiert, oder f) die Nukleotidsequenz der SEQ ID NO: 6 oder eine zu der Nukleotidsequenz der SEQ ID NO: 6 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO: 6 oder einer zu der Nukleotidsequenz der SEQ ID NO: 6 komplementären Nukleotidsequenz hybridisiert .e) the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4 or hybridized with the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4, or f) comprises the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6 or hybridizes with the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6.
g) die Nukleotidsequenz der SEQ ID NO : 7 oder eine zu der Nukleotidsequenz der SEQ ID NO : 7 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO: 7 oder einer zu der Nukleotidsequenz der SEQ ID NO : 7 komplementären Nukleotidsequenz hybridisiert .g) comprises the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7 or hybridizes with the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7.
h) die Nukleotidsequenz der SEQ ID NO: 8 oder eine zu der Nukleotidsequenz der SEQ ID NO : 8 komplementäre Nukleotidsequenz umfaßt oder mit der Nukleotidsequenz der SEQ ID NO : 8 oder einer zu der Nukleotidsequenz der SEQ ID NO: 8 komplementären Nukleotidsequenz hybridisiert .h) comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 8 or hybridizes with the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 8.
4. Vektor oder mobiles genetisches Element, enthaltend mindestens eine DNA-Sequenz gemäß Anspruch 3 und/oder mindestens eine der DNA-Sequenz entsprechende RNA-Sequenz - ΛY-4. Vector or mobile genetic element containing at least one DNA sequence according to claim 3 and / or at least one RNA sequence corresponding to the DNA sequence - ΛY-
und/oder mindestens eine aus DNA- und RNA-Nukleotiden zusammengesetzte Mischsequenz.and / or at least one mixed sequence composed of DNA and RNA nucleotides.
5. Vektor oder mobiles genetisches Element nach Anspruch 4, dadurch gekennzeichnet, daß die DNA-Sequenz und/oder die der DNA-Sequenz entsprechende RNA-Sequenz und/oder die Mischsequenz in Antisense-Orientierung enthalten ist.5. Vector or mobile genetic element according to claim 4, characterized in that the DNA sequence and / or the RNA sequence corresponding to the DNA sequence and / or the mixed sequence is contained in antisense orientation.
6. Vektor oder mobiles genetisches Element nach Anspruch 4 oder 5, dadurch gekennzeichnet, daß die RNA-Sequenz in einem RNA-Doppelstrang enthalten ist.6. Vector or mobile genetic element according to claim 4 or 5, characterized in that the RNA sequence is contained in an RNA double strand.
7. Eukaryotische oder prokaryotische Wirtszelle, enthaltend mindestens eine DNA-Sequenz gemäß Anspruch 3 und/oder eine der DNA-Sequenz entsprechende RNA-Sequenz und/oder mindestens eine aus DNA- und RNA-Nukleotiden zusammengesetzte Mischsequenz .7. Eukaryotic or prokaryotic host cell containing at least one DNA sequence according to claim 3 and / or an RNA sequence corresponding to the DNA sequence and / or at least one mixed sequence composed of DNA and RNA nucleotides.
8. Eukaryotische oder prokaryotische Wirtszelle nach Anspruch 7, dadurch gekennzeichnet, daß die DNA-Sequenz und/oder die der DNA-Sequenz entsprechende RNA-Sequenz und/oder die Mischsequenz in Antisense-Orientierung enthalten ist.8. Eukaryotic or prokaryotic host cell according to claim 7, characterized in that the DNA sequence and / or the RNA sequence corresponding to the DNA sequence and / or the mixed sequence is contained in an antisense orientation.
9. Eukaryotische oder prokaryotische Wirtszelle nach Anspruch 7 oder 8, dadurch gekennzeichnet, daß die RNA-Sequenz in einem RNA-Doppelstrang enthalten ist.9. Eukaryotic or prokaryotic host cell according to claim 7 or 8, characterized in that the RNA sequence is contained in an RNA double strand.
10. Pflanze oder Teile davon, transformiert mit mindestens einer DNA-Sequenz nach Anspruch 3 und/oder mindestens einer der DNA-Sequenz entsprechenden RNA-Sequenz und/oder mindestens einer aus DNA- und RNA-Nukleotiden zusammengesetzten Mischsequenz .10. Plant or parts thereof, transformed with at least one DNA sequence according to claim 3 and / or at least one RNA sequence corresponding to the DNA sequence and / or at least one mixed sequence composed of DNA and RNA nucleotides.
11. Transgene Pflanze nach Anspruch 10, dadurch gekennzeichnet, daß die Pflanze mit der DNA-Sequenz und/oder der RNA- - A $ -11. Transgenic plant according to claim 10, characterized in that the plant with the DNA sequence and / or the RNA - A $ -
Sequenz und/oder der Mischsequenz in Antisense-Orientierung transformiert ist.Sequence and / or the mixed sequence is transformed into an antisense orientation.
12. Transgene Pflanze nach Anspruch 10 oder 11, dadurch gekennzeichnet, daß die RNA-Sequenz in einem RNA- Doppelstrang enthalten ist.12. Transgenic plant according to claim 10 or 11, characterized in that the RNA sequence is contained in an RNA double strand.
13. Samen von Pflanzen nach einem der Ansprüche 10 bis 12.13. Seeds of plants according to one of claims 10 to 12.
14. Verwendung einer DNA-Sequenz nach Anspruch 3 und/oder einer der DNA-Sequenz entsprechenden RNA-Sequenz zur Herstellung einer transgenen Pflanzenzelle oder Pflanze mit einer gegenüber dem Wildtyp erhöhten Expression der codierenden Region des Aminosäuretransporters .14. Use of a DNA sequence according to claim 3 and / or an RNA sequence corresponding to the DNA sequence for producing a transgenic plant cell or plant with an increased expression of the coding region of the amino acid transporter compared to the wild type.
15. Verwendung einer DNA-Sequenz nach Anspruch 3 und/oder einer der DNA-Sequenz entsprechenden RNA-Sequenz in Antisense- Orientierung zur Herstellung einer transgenen Pflanzenzelle oder Pflanze mit einer gegenüber dem Wildtyp verminderten Expression der codierenden Region des Aminosäuretransporters .15. Use of a DNA sequence according to claim 3 and / or an RNA sequence corresponding to the DNA sequence in antisense orientation for the production of a transgenic plant cell or plant with a reduced expression compared to the wild type of the coding region of the amino acid transporter.
16. Verwendung nach Anspruch 15 zur Herstellung einer transgenen. Pflanze mit einer gegenüber dem Wildtyp verminderten Fähigkeit zur Einlagerung von organischen Stickstoffverbindungen, insbesondere Glutamin, Asparagin, Citrullin, Gamma-Aminobuttersäure und Auxin, in Speicherorganen .16. Use according to claim 15 for the production of a transgenic. Plant with a reduced ability compared to the wild type to store organic nitrogen compounds, in particular glutamine, asparagine, citrulline, gamma-aminobutyric acid and auxin, in storage organs.
17. Verwendung nach Anspruch 15 oder 16 zur Herstellung einer transgenen Pflanze mit einer gegenüber dem Wildtyp erhöhten Speicherfähigkeit für Speicherstoffe. 17. Use according to claim 15 or 16 for the production of a transgenic plant with an increased storage capacity for storage substances compared to the wild type.
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