EP1276882A2 - Procede de modification genetique d'une plante - Google Patents

Procede de modification genetique d'une plante

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Publication number
EP1276882A2
EP1276882A2 EP01905823A EP01905823A EP1276882A2 EP 1276882 A2 EP1276882 A2 EP 1276882A2 EP 01905823 A EP01905823 A EP 01905823A EP 01905823 A EP01905823 A EP 01905823A EP 1276882 A2 EP1276882 A2 EP 1276882A2
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EP
European Patent Office
Prior art keywords
nucleic acid
saccharide
sucrose
sut2
coding
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.)
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Application number
EP01905823A
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German (de)
English (en)
Inventor
Wolf-Bernd Frommer
John M. Ward
Andreas Weise
Laurence Barker
Waltraud Schulze
Christina KÜHN
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.)
FROMMER WOLF BERND PROF DR
Original Assignee
FROMMER WOLF BERND PROF DR
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Priority claimed from DE10050233A external-priority patent/DE10050233A1/de
Application filed by FROMMER WOLF BERND PROF DR filed Critical FROMMER WOLF BERND PROF DR
Publication of EP1276882A2 publication Critical patent/EP1276882A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to nucleic acid molecules which encode a saccharide, in particular sucrose transporter, vectors and host cells which contain these nucleic acid molecules, and to fungi, plant cells and plants transformed with the nucleic acid described and vectors transformed.
  • the invention further relates to methods for modifying the transport of saccharide, in particular sucrose, in plants.
  • Higher plants have heterotrophic tissues that are supplied with carbohydrates by autotrophic tissues.
  • the main form of transport of the carbohydrates is sucrose and its derivatives.
  • the heterotrophic tissues are supplied via the phloem, which the organs, which photoassimilates, ie carbohydrates, provide in excess and which can export them, so-called source organs with organs who have to import photoassimilates in their net balance, so-called sink organs.
  • Source organs are, for example, adult leaves and germinating seeds.
  • Sink organs are, for example, young leaves, young tubers, roots, fruits, flowers and other reproductive organs.
  • the phloem is made up of various cell types such as sieve elements, escort cells, parenchyma cells and bundles vaginal cells built up.
  • the translocation current of the photoassimilates moves from places of export to places of import.
  • Both the loading of the phloem with photoassimilations and the unloading can in principle be carried out in an apoplastic and symplastic way, with a large number of factors such as osmotic conditions, concentration gradients, plasma membranes to be passed, etc. influencing the loading and unloading.
  • Both the loading of the phloem and the supply of the sink organs with photoassimilates are accordingly highly complex processes, which obviously involve a large number of closely linked and regulated transport steps. These transport steps take place with the participation of different plasma membrane proteins, in particular transport proteins. So z. B.
  • sucrose transporter sucrose transporter
  • the SUT genes encode hydrophobic proteins that have 12 transmembrane domains and can be clearly distinguished from the members of the hexose transporter family. London et al. (The Plant Cell (1999) 11, 707-726) suggests that the members of the SUT family have a high affinity for sucrose. It is assumed that the sucrose transporter SUT1 from Lycopersicon esculentum, Nicotiana tabacum and Solanum tuberosu is responsible for long-distance transport in the phloem (Riesmeier et al., Plant Cell (1993) 5, 1591-1598).
  • WO94 / 00574 discloses the DNA and amino acid sequences of SUT1 from spinach and potato. This one in The sucrose transporter located in the plasma membrane of the sieve elements of the phloem is an essential component for the long-distance transport of sucrose in the phloem, as can be seen, for example, in antisense inhibition experiments in transgenic potato and tobacco plants (Riesmeier et al., EMBO J (1994) 13, 1-7) (Lalonde et al., Loc. Cit.). The expression pattern of SUT1, in particular the expression in the entire phloem, proves that SUT1 is responsible for maintaining the concentration gradient of sucrose between loading and unloading zones (leaves, respectively sink organs).
  • SUT1 therefore appears to be less responsible for the (initial) loading of the phloem in source organs than for the return of sucrose that has migrated from the phloem. This function is further confirmed by the relatively high affinity and the associated low transport capacity. SUT1 can therefore import very small amounts of sucrose from the apoplast into the phloem (back) and thus keeps the apoplastic concentrations low. Since he works at these low concentrations, he cannot be responsible for high transport rates.
  • sucrose transporters in addition to the sucrose transporters, other functional elements are involved in the sucrose transport, for example regulator and sensor elements.
  • regulator and sensor elements the extraordinary complexity of the sucrose transport system has so far not allowed a specific assignment of structures to functions and a prediction of their interactions in a manner that makes it possible to carry out targeted interventions in the saccharide, in particular sucrose transport with the aim of certain properties to obtain improved plants. This can be seen most clearly from the fact that an overexpression of SUT1 did not result in any changes in the allocation of assimilates, but only showed an influence on the flowering behavior (US 6,025,544; P 44 39 748.8).
  • the technical problem on which the present invention is based is therefore methods and means for the production of a genetic engineering provide modified plant that allow targeted intervention in the saccharide, especially sucrose transport streams of the plant such that a plant with improved properties, e.g. B. higher sugar levels in sink organs, especially in the harvesting organs can be generated.
  • the invention solves this problem by providing a method for modifying the saccharide flux and / or the saccharide concentration, in particular sucrose flux and / or concentration in tissues of a plant, according to which a modified activity of a saccharide transporter in a plant with low saccharide affinity but high saccharide transport capacity.
  • the teaching according to the invention provides means and methods for specifically influencing the saccharide flux and the saccharide concentration in the various tissues of the plant by modifying the activity of a saccharide transporter with low saccharide affinity and high saccharide transport capacity to modify ie, wherein • this method produced a modified plant in the course.
  • the present invention provides for the first time an HCS system, that is to say a sucrose transport system with a high transport capacity for sucrose but low affinity for sucrose, which is expressed, in particular in microveins of a plant.
  • a modification of the activity of the sacchar ridtransports is understood to mean a change in the normally present activity compared to the wild-type activity, eg. B. Complete suppression, reduction, or increased activity.
  • This increase in activity may be due to a change in the activity of the protein itself.
  • B. by post-transcriptional modifications such as phosphorylations or dephosphorylations.
  • it can also be due to a changed expression rate of the coding gene, a changed stability or translation rate of the mRNA formed or the like, ie also to a changed amount of saccharide transporters present in a tissue.
  • the activity of the saccharide transporter can also be modified by the activity of an element which controls or regulates the activity of the saccharide transporter, for example a sensor or regulator protein.
  • a saccharide means sucrose and a saccharide transporter means a sucrose transporter, in particular, unless stated otherwise, SUT4 (sucrose transporter 4), that is to say a transporter with low affinity for sucrose and a high transport capacity for sucrose.
  • SUT4 sucrose transporter 4
  • Low or low affinity in the sense of the present invention is an affinity which is below the SUT1 affinity, for example 50%, preferably 80%, 100%, 200%, 300% or 400% below that of SUT1, for example an affinity K m > 2.7 mM, preferably> 4 mM,> 6 mM,> 8 mM,> 10 mM,> 15 mM,> 20 mM and preferably> 25, in particular 26 mM.
  • High transport capacity in the sense of the present invention is a transport capacity which is above the transport capacity of SUT1, for example 50%, 100%, 150%, 200%, 300%, 400% or 500% above that of SUT1.
  • a transport capacity V m ⁇ of> 0.7, in particular>1,>1.5,>2,>3,> 3.5 in particular 3.6 n ol cm -2 in ⁇ 1 is preferred.
  • the invention is based on the knowledge and the teaching derived therefrom, using technical, in particular genetic engineering, means to influence an activity of the saccharide transporter mentioned, which may only be endogenous, or to introduce such activity into a plant.
  • sink organs is understood to mean organs or tissues of plants which have to import photoassimilates in their net balance.
  • plant organs or tissues are young leaves, tubers, fruits, roots, flowers, reproductive organs, wood, marrow tissue, buds, seeds, beets etc.
  • source organ is understood to mean organs or tissues of plants which have an excess of photoassimilates in their net balance and can export them.
  • Source organs are e.g. B. adult leaves and germinating seeds, tubers and onions.
  • an increased expression rate of the saccharide transporter modified according to the invention or an increased transport rate, for example in transfer cells and / or sieve elements considerably increases the saccharide, in particular sucrose, loading of the phloem, in particular under high light intensity or CO 2 concentration, as a result of which plants with harvesting organs which have an increased sucrose content can advantageously be obtained.
  • the invention thus solves the problem in particular by providing a method for modifying the saccharide flux and / or the saccharide concentration in the tissues of a plant, the activity of a saccharide transporter being modified with low affinity for the saccharide and high transport capacity for the saccharide by transforming at least one plant cell with at least one vector which contains nucleotide sequences which enable the modification of the saccharide transporter, and the plant cell which contains these nucleotide sequences in a stably integrated manner is regenerated into a plant in whose tissue a modified saccharide flux and / or a modified one is regenerated Saccharide concentration, in each case in comparison with a wild-type plant, that is to say a plant not transformed according to the invention, is present.
  • the invention thus provides the teaching of specifically modifying a saccharide transporter with a high transport capacity for the saccharide and with a low affinity for the saccharide, this being done by means of genetic engineering manipulation of the plant.
  • this can be done by transforming a plant cell with at least one vector containing the coding nucleotide sequences of the saccharide transporter, which preferably allows over-expression of the saccharide transporter in transformed tissue after stable integration of the nucleotide sequences into the genome of the transformed cell.
  • Plants produced by the procedure according to the invention have e.g. B. a higher carbohydrate content in sink organs, such as roots, fruits, bulbs, flowers or seeds.
  • sink organs such as roots, fruits, bulbs, flowers or seeds.
  • there is a higher carbohydrate content especially in harvesting organs of the plant, which are often sinking organs.
  • an increased oil content can be found in the harvesting organs.
  • the observation that the number of harvesting organs per plant can be increased and their weight can be increased is particularly advantageous.
  • the overexpression of the saccharide transporter of the type mentioned in source organs provided according to the invention can advantageously also result in the flowering time of the transformed plant being changed. Since the transport of sugar in the phloem is often reciprocally coupled with the amino acid transport in the phloem, the increase in the saccharide content in the phloem provided according to the invention can cause the undesired amino acid content in sink organs, e.g. B. potato tubers or tap roots of the sugar beet are lowered. Finally, the procedure according to the invention enables the glycosylation rate to be endogenous to substances present in plants or also to exogenously administered substances such as xenobiotics, eg B. herbicides or pesticides, to increase their mobility.
  • xenobiotics eg B. herbicides or pesticides
  • the above-described overexpression in source organs is achieved by cloning SUT4-encoding nucleotide sequences into a vector under the control of source-specific promoters, that is to say in particular leaf-specific and / or guide cell-specific promoters at least one plant cell is transformed and, preferably stably, integrated into the genome of the plant cell.
  • constitutively expressing promoters such as the 35SCaMV promoter, the control cell-specific rolC promoter from Agrobacterium or the enhanced PMA4 promoter (Morian et al., Plant J (1999) 19, 31-41 ) to use.
  • the invention provides for the modification of the saccharide transport to be carried out by the saccharide transporter is modified by overexpression in leaf mesophyll tissue and / or leaf epidermis.
  • the specific expression of SUT4-coding nucleotide sequences in these tissues leads to a competitive effect with regard to the endogenous sucrose transporter active in the sieve elements, so that the carbohydrate content in the leaves is increased.
  • the plants produced in this way have larger leaves, which moreover have an improved protection potential against pathogens.
  • genes with a defense function are activated by an increased sugar content.
  • leaf mesophyll and epidermis provided in this preferred embodiment can be achieved by the use of the StLSl / L700 promoter (Stockhaus et al., Plant Cell (1989) 1, 805-813), other epidermis-specific promoters or that provided in a particularly preferred embodiment PFP promoter (palisade parenchyma) (WO 98/18940) for expressing the SUT4-coding nucleotide sequences can be achieved.
  • a further preferred embodiment of the present invention provides for overexpression of the saccharide transporter to be provided specifically in sink cells or organs, in particular developing seeds of a plant.
  • this enables an improved germination rate to be achieved, since both the carbohydrate and especially the oil content of the seeds is increased.
  • tissue-specific promoters to be used preferably in this embodiment for the expression of the SUT4-coding nucleotide sequences in seed tissue are e.g. the Vicilin promoter from Pisum sativum (Newbigin et al., Planta (1990) 180, 461-470).
  • an aforementioned method is provided, overexpression of the saccharide transporter being provided by using tissue-specific regulatory elements for the epidermis and parenchyma of sink organs.
  • the increased expression of the saccharide transporter used according to the invention in sink organs increases their saccharide absorption capacity and the saccharide flux into the sink tissue.
  • Plants produced according to the invention therefore have, for example, larger, more colorful and / or a higher number of flowers, larger seeds, larger tubers or larger tap roots.
  • the sink organs can also be characterized by a higher carbohydrate and oil content, an improved structure, in particular strength, faster growth and / or improved cold tolerance, if necessary, due to the higher content of osmotically active substances. It is also possible to influence the flowering time and duration as well as the development of the fruit.
  • the invention provides for the above-mentioned expression in the sink epidermis and parenchyma the AAP-1 (amino acid permease 1) promoter, for example the A-rabidopsis promoter AtAAPl (expression in endosperm and during early embryo development) or AtAAP2 (expression in phloem of the funiculus) (Hirner et al. , Plant J. (1998) 14, 535-544, the B33 (patent) promoter (Rocha-Sosa et al., EMBO J.
  • AAP-1 amino acid permease 1 promoter
  • AtAAPl expression in endosperm and during early embryo development
  • AtAAP2 expression in phloem of the funiculus
  • the invention also relates to the modification of the saccharide transport activity in the tissues of a plant, the activity of a saccharide transporter, in particular sucrose transporter, having a high transport capacity for sucrose and a low affinity for sucrose being suppressed or reduced, in particular inhibited or cosuppressed becomes.
  • the activity of the saccharide transport can be suppressed or reduced by transforming plant cells with vectors, the nucleotide sequences coding for the sacc arid transporter used according to the invention or parts thereof sufficient for anti-sense repression in anti - have sense orientation to a promoter and are preferably stably integrated into the genome of the plant cell.
  • the expression of this antisense RNA suppresses or reduces the formation of the endogenously present saccharide transporter mentioned, so that the saccharide flux caused by this transporter can be manipulated.
  • the activity of this saccharide transporter is reduced or suppressed by the suppression effects introduced into the plant.
  • several copies of a vector are introduced into at least one plant cell, in particular stably in its genome, which has the nucleotide sequences coding for the saccharide transporter or parts thereof, and these copies being integrated in the genome.
  • sucrose transporter by RNA double-strand inhibition.
  • the aforementioned techniques for suppressing or reducing the activity of the saccharide transporter with low affinity for sucrose but high sucrose transport capacity are used, for example, in source tissues, e.g. B. leaves to build a higher carbohydrate content, especially sucrose.
  • this can be done by reducing or suppressing the saccharide transport capacity in source organs as described above. This reduces the structure and strength of sink organs, while the sucrose. Carbohydrate content in source organs increases.
  • provision can be made to suppress or reduce the activity of the saccharide transporter in the guard cells, for example by mutagenesis of endogenously present nucleotide sequences which code in the guard cells and which code for the saccharide transporter, by cosuppression effects, RNA double-strand inhibition or by using antisense constructs.
  • the capacity to open or close the stomata can be changed, in particular increased or reduced, by modifying saccharide transport processes and the associated changes in the provision of energy and osmotically active substances in guard cells.
  • a higher opening rate of the stomata allows an improved supply for C0 2 , so that the photosynthesis rate is improved.
  • a vector is used in order to achieve the aforementioned effects, in which the nucleotide sequences coding for the saccharide transporter used according to the invention are in sense or antisense orientation, e.g. B. under the control of a guard cell-specific promoter, e.g. B. the KATl promoter (Nakamura et al., Plant Physiol. (1995) 109, 371-374).
  • the saccharide transporter SUT4 is also expressed in sink organs, provision can be made to reduce the saccharide import into the sink cells or organs or preferably into certain sink cells or organs, particularly preferably into the flower. This can cause carbohydrates to accumulate in source cells or organs, which are useful for other synthetic routes, and the relative activity of individual sink cells can be shifted in favor of others, thus improving the yields qualitatively and quantitatively.
  • nucleotide sequences coding for SUT4 in particular sucrose transporters, particularly preferably SUT4-coding nucleotide sequences, further nucleotide sequences are used for the transformation which are used in the there is a functional connection with the sucrose concentration and the sucrose flux in the tissues of a plant.
  • these are nucleotide sequences which encode SUT1 or SUT2, e.g. B. genomic or cDNA nucleotide sequences.
  • SUT1 genomic and cDNA sequences are e.g. B. from WO94 / 00574 (potato, spinach), Riesmeier et al. , a. a. 0. (potato), Riesmeier et al. , (EMBO J. (1992) 11, 4705-4713 (spinach)), Bürkle et al. .
  • SUT1-coding nucleotide sequences used according to the invention as well as the amino acid sequence derived therefrom, shown in SEQ ID NOS 22 and 23, are also the subject of the present invention.
  • SUTl represents a sucrose transporter with high affinity for sucrose but low transport capacity for sucrose. Coexpression of sucrose transporters with different affinities for sucrose in one and the same tissue, eg in sieve elements, allows a regulated and the actual one present in the plant Manipulation of the sucrose flux that meets the circumstances.
  • a vector for transforming the at least one plant cell which has nucleotide sequences coding for SUT2.
  • SUT2 acts in particular as a regulator and sensor.
  • SUT2 also exhibits the biological activity of a low-affinity sucrose transport.
  • SUT2's transport rates are also low.
  • SUT2 is a regulator and / or sensor of the sucrose transporter, which in particular can determine its own transport activity and, if necessary, mediate it further. Its transport activity can on the one hand be a functional component of its sensor activity and on the other hand its sensor activity can be seen as a functional component of its transport activity.
  • SUT2 with its low affinity for sucrose, is a flux sensor that possibly transports a substrate, namely sucrose, and uses a signal cascade or is a component thereof, which measures the rate of transport.
  • the affinity of SUT2 for sucrose is lower than that of SUT4.
  • the N-termini of proteins of the SUT / SUC gene family impart a modified affinity for their substrate, in particular sucrose.
  • the N-termini of SUT2 but also of SUT1 mediate a modified sucrose affinity, in the case of SUT2 low affinity for sucrose and in the case of SUT1 high affinity.
  • the invention therefore also relates to the use of N-termini from sucrose transporters or nucleotide sequences encoding them, in particular vegetable sucrose transporters for modifying the sucrose transport or the sucrose sensing in plants, in particular for producing modified sucrose transporters and sensors modified affinity for sucrose in plants.
  • the invention also relates to the use of SUT2 and / or SUT2-coding DNA sequences, in particular the SUT2 loop as a regulator and sensor of sucrose transport, in particular for regulating the SUT4 and / or SUT1 activity, for example in plant or fungal cells.
  • SUT2 can be induced by sucrose.
  • SUT2 regulates the relative activity of in the same cell type, e.g. B. in the sieve elements, existing sucrose transporters.
  • SUT2 regulates the activity of the sucrose transporter SUT1, which has high sucrose affinity but low transport capacity and the SUT4 sucrose transporter with high transport capacity but low sucrose affinity. This regulation can be done by controlling expression, protein activity, e.g. B. by protein modification or the turnover rate of mRNA or protein and result in an increase or decrease in activity.
  • SUT2 is expressed in plants, particularly in the large leaf veins of adult leaves, flowers and sink organs.
  • the invention therefore also relates to the aforementioned N-termini and central loops or loops of proteins from the SUT / SUC gene family, in particular of SUT1, SUT2 and / or SUT4, and the nucleotide sequences encoding these regions. In a preferred embodiment, these are shown in SEQ ID No. 24, 25 and 26.
  • the N-termini of LeSUT2 (Lycopersicon esculentum) and StSUT2 (Solanum tuberosum) comprise the first 62 amino acids of the protein and are encoded by nucleotides 1 to 186 of SEQ ID No. 4 (Lycopersicon esculentum) and No. 29 (Solanum tuberosum).
  • the central loop of LeSUT2 is encoded by nucleotides 844 to 1131 of SEQ ID No. 4 and of StSUT2 by nucleotides 847 to 1134 of SEQ ID No. 29.
  • the SUT1, SUT4 and / or SUT2 coding sequences preferably arranged in a vector, in sense or antisense orientation to at least one regulatory element, in particular a promoter, e.g. B.
  • a promoter e.g. B.
  • one of the aforementioned promoters can be transformed into plant cells, the activity of cotransformed and / or endogenously present SUT4 being modified after integration in the genome and expression of the product.
  • this relates to a aforementioned method, in which a vector is transformed into the plant cell which contains nucleotide sequences which encode SUT2, preferably in sense or antisense orientation, under the operative control of a regulatory element, in particular a promoter. It can be provided that the vector containing the SUT2-encoding nucleotide sequences without further vectors, the z. B. SUT4 or SUTl-encoding nucleotide sequences to transform.
  • SUT2 is in particular a sucrose concentration sensor and regulator with the above-mentioned transporter properties as well as a sucrose flux sensor and regulator in the transformed plant, in particular the endogenously present sucrose transporter, namely SUT4 and / or SUTl.
  • This re- Gulation can take place at the transcriptional or post-transcriptional level, for example by direct protein interaction or indirectly via signal transduction.
  • the invention also relates to the use of the SUT2-encoding nucleotide sequences or parts thereof, in particular the nucleotide sequence encoding the N-terminal protein region, for transforming plant cells, which can be transformed together with SUT1 and / or SUT4-encoding nucleotide sequences , Even in such a case, the overexpression, cosuppression or antisense repression of SUT2 can modify the activity of SUT1 and / or SUT4, e.g. B. increase or decrease.
  • parts of the SUT2-coding nucleotide sequences in particular the nucleotide sequences of SUT2 (SEQ ID No.
  • SUT2 nucleotide sequences which contain SUT2 or parts of SUT2 are also referred to in the context of the present invention as modified SUT2 nucleotide sequences.
  • SUT2 interacts with other proteins, in particular with regulators, signal transduction factors and other sucrose transporters. Therefore, using SUT2 by interaction cloning, further regulators can be identified for whom protection is also sought.
  • the invention also relates to, preferably isolated and purified, regulatory proteins and sensor proteins, as well as nucleotide sequences coding for them, which contain the central cytoplasmic loop of SUT2, in particular chimeric proteins and nucleic acids with N- and C-terminal regions of other sucrose transporters, or nucleotide sequences coding for them, wherein these chimeric proteins or nucleic acids contain the central loop of SUT2.
  • the central cytoplasmic loop or loop has a biological activity as a regulator element and / or sensor and / or signal transducer.
  • the invention therefore relates to the aforementioned methods for modifying the activity of a sucrose transporter with low affinity for sucrose but high transport activity for sucrose in the context of which known or modified SUT4, SUT1 and / or SUT2-coding nucleotide sequences are used to achieve the modification and to obtain an improved transgenic plant.
  • the invention therefore also relates to methods for producing transgenic, modified plants which are modified Have activity of said sucrose transporter and, preferably stably integrated in the genome, contain modified SUT1, SUT2 and / or SUT4 nucleotide sequences.
  • the invention also relates to the transgenic plants, plant cells, organs or parts of organs and plants thus produced which are distinguished by the changed activity of the sucrose transporter mentioned and at least one of the nucleotide sequences mentioned selected from the group consisting of the nucleotide sequences, in particular genes for saccharide transporters, such as SUT and SUC genes, preferably for SUT1; SUT2; SUT4; SUT1 and SUT2; SUTl and SUT4; SUT2 and SUT4; SUTl and SUT2 and SUT4 included in a modified form.
  • the nucleotide sequences selected from the group consisting of the nucleotide sequences, in particular genes for saccharide transporters, such as SUT and SUC genes, preferably for SUT1; SUT2; SUT4; SUT1 and SUT2; SUTl and SUT4; SUT2 and SUT4; SUTl and SUT2 and SUT4 included in a modified form.
  • a modified nucleotide sequence is understood to mean a nucleotide sequence which differs from the wild-type sequence, in particular the wild-type gene, for example a deviation which is based on nucleotide insertions, inversions, deletions, exchanges, additions or the like.
  • modified nucleotide sequences also represent those genes which contain the coding nucleotide sequence of the wild-type, this coding nucleotide sequence being operatively in the sense or antisense orientation with a heterologous promoter, e.g. B. are linked to a tissue-specific or constitutively expressing promoter.
  • a modified nucleotide sequence can also be present if it corresponds exactly to the wild-type sequence, but is present in an additional copy number and / or at a different location in the genome than the naturally occurring sequence.
  • a modified nucleotide sequence is also present if the naturally endogenously occurring nucleotide sequence has been changed by mutagenesis, for example transposon mutagenesis.
  • modified genes are understood to mean those nucleotide sequences which have deviations in the nucleotide sequence of their regulatory and / or protein-coding regions, e.g. B.
  • Modified nucleotide sequences or modified genes can also be chimeric nucleotide sequences or genes, for example those protein-coding regions which are composed of two or more different naturally occurring nucleotide sequences, for example constructs which encode sequences as N-terminal nucleotide sequence SUT2 (SEQ ID No. have. 24) and as a central and 3 ⁇ -terminal region SUTl-coding sequences.
  • modified genes are but understood as 5 X - coding region sequences of the SUTl gene (for example, SEQ ID NO. 25) and a central portion o- the / 3 and region sequences of the SUT2 gene contained.
  • Modified genes can accordingly z. B. the wild-type coding sequences and heterologous promoters z. B. from other organisms or from other genes.
  • a gene is understood to mean a protein-coding nucleotide sequence which is under the operative control of at least one regulatory element.
  • the invention also relates to agents for modifying the transport of sucrose.
  • agents for modifying the transport of sucrose are nucleic acid molecules encoding a saccharide transporter with low saccharide affinity and high transport capacity for the saccharide or parts thereof, in particular sucrose, selected from the group consisting of
  • nucleic acid molecules which comprise the nucleotide sequence shown under SEQ ID No. 1, 2 or 27, a part or a complementary strand thereof, b) nucleic acid molecules which encode a protein with the amino acid sequence shown under SEQ ID No. 5, 6 or 28 and c) nucleic acid molecules which hybridize with one of the nucleic acid molecules mentioned under a) and b).
  • the saccharide transporter is a sucrose transporter, in particular SUT4, e.g. B. from Arabidopsis (Arabidopsis thaliana, At), tomato (Lycopersicon esculentum, Le) or potato (Solanum tuberosum, St).
  • SUT4 a sucrose transporter
  • the aforementioned nucleic acid molecules are also referred to as SUT4 coding sequences.
  • the invention also relates to nucleic acid molecules encoding a sensor and / or regulator for the sucrose transport in plants and with the properties of a low-affinity sucrose transporter with low transport rates or parts thereof, selected from the group consisting of
  • nucleic acid molecules which comprise the nucleotide sequence shown under SEQ ID No. 3, 4, 24, 26 or 29, a part or a complementary strand thereof, b) Nucleic acid molecules which contain a protein with the one under SEQ ID No. 7, 8 or 30 amino acid sequence shown encode and c) nucleic acid molecules that hybridize with one of the nucleic acid molecules mentioned under a) and b).
  • the saccharide sensor and / or regulator is a sucrose sensor and / or regulator, in particular SUT2, e.g. from potato, tomato or arabidopsis.
  • SUT2 a sucrose sensor and / or regulator
  • the aforementioned nucleic acid molecules are also referred to as SUT2 coding sequences.
  • nucleic acid molecules according to the invention or used according to the invention can be isolated and purified from natural sources, preferably from the potato, or they can be synthesized by known processes. Using common molecular biological techniques, it is possible to insert different types of mutations into the inventive or the already known insert inserted nucleic acid molecules, which results in the synthesis of proteins with possibly changed biological properties, which are also covered by the invention. Mutations in the sense of the invention also concern all deletion mutations which lead to shortened proteins. Other molecular mechanisms such as insertions, duplications, transpositions, gene fusion, nucleotide exchange as well as gene transfer between different microorganism strains and others can lead to modifications of the activity and / or regulation of the protein, for example.
  • mutant proteins which have an altered transport capacity or sucrose affinity and / or which are no longer subject to the regulatory mechanisms normally present in the cells or in an altered form.
  • mutant proteins according to the invention can be produced which have a changed stability, substrate specificity or a changed effector pattern or a modified activity, temperature, pH value and / or concentration profile.
  • teaching according to the invention relates to proteins which have a changed active protein concentration, pre- and post-translational modifications, for example signal and / or transport peptides and / or other functional groups.
  • the invention also relates to nucleic acid molecules which hybridize with the aforementioned nucleic acid molecules according to the invention.
  • hybridization means hybridization and conventional hybridization conditions, as described in Sambrook et al. (Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd edition, 1989), preferably under stringent conditions.
  • one speaks of a hybridization if, after washing for 15 minutes with 2 ⁇ SSC and 0.1% SDS at 52 ° C., preferably at 60 ° C. and particularly preferably at 65 ° C., in particular for 15 minutes in 0, 5 x SSC and 0.1% SDS at 52 ° C, preferably at 60 ° C and particularly preferably at 65 ° C a positive hybridization signal is observed.
  • a nucleotide sequence which hybridizes under such washing conditions with one of the nucleotide sequences specified in the sequence listing is a nucleotide sequence according to the invention.
  • nucleic acid molecules can be identified and isolated using the nucleic acid molecules according to the invention or parts of these molecules or the complementary strand.
  • nucleic acid molecules can be used as the hybridization sample which have exactly or essentially the nucleotide sequences shown under SEQ ID No. 1, 2, 3 or 4 or parts of this sequence.
  • the fragments used as the hybridization sample can also be synthetic fragments which are produced using conventional synthesis techniques and whose sequence essentially corresponds to that of a nucleic acid molecule according to the invention.
  • the molecules hybridizing with the nucleic acid molecules according to the invention also include fragments, derivatives and al lelic variants of the nucleic acid molecules described above, which encode a protein according to the invention.
  • “Fragments” are understood to mean parts of the nucleic acid molecules that are long enough to encode the protein described.
  • the term “derivative” in the context of the invention means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or more positions, but have a high degree of homology to these sequences.
  • Homology means a sequence identity of at least 70%, in particular an identity of at least 75%, preferably over 80% and particularly preferably over 90%, 95%, 97% or 99% at the nucleic acid level.
  • the proteins encoded by these nucleic acid molecules have a sequence identity to the amino acid sequence given in SEQ ID No.
  • the deviations from the nucleic acid molecules described above may have arisen, for example, through deletion, substitution, insertion or recombination.
  • These can be both naturally occurring variations, for example sequences from other organisms, or mutations, whereby these mutations can have occurred naturally or have been introduced by targeted mutagenesis (UV or X-rays, chemical agents or others).
  • the variations can be synthetically produced sequences.
  • the allelic variants can be both naturally occurring variants yours, as well as synthetically produced or recombinant DNA techniques.
  • the proteins encoded by the different variants of the nucleic acid molecules according to the invention have certain common characteristics, such as activity, active protein concentration, post-translational modifications, functional groups, molecular weight, immunological reactivity, conformation and / or physical properties, such as running behavior in gel electrophoresis, chromatographic behavior, sedimentation coefficient , Solubility, spectroscopic properties, stability, pH optimum, isoelectric pH, temperature optimum and / or others.
  • the nucleic acid molecules according to the invention can be both DNA and RNA molecules.
  • DNA molecules according to the invention are, for example, genomic DNA or cDNA molecules.
  • the invention further relates to vectors which contain nucleic acid molecules according to the invention.
  • vectors can e.g. B. plasmids, liposomes, cosmids, viruses, bacteriophages, shuttle vectors and other vectors common in genetic engineering.
  • the vectors can also have further functional units which bring about or contribute to stabilization and / or replication of the vector in a host organism.
  • the vectors are also detected according to the invention in which the nucleic acid molecule contained therein is operatively linked to at least one regulatory element which ensures the transcription and synthesis of translatable nucleic acid molecules in pro- and / or eucaryotic cells.
  • regulatory elements can be promoters, enhancers, operators and / or transcription termination signals.
  • the vectors can also antibiotic resistance genes, herbicide resistance genes z. B. selection markers included.
  • the invention also relates to the aforementioned vectors, which in addition to the nucleic acid sequences which are under the control of at least one regulatory element and which encode the SUT4 and / or SUT2 according to the invention, also comprise a nucleic acid sequence which is likewise under the control of at least one regulatory element, encoded the SUTl.
  • a vector thus has the genetic information for at least two proteins involved in the transport of sucrose.
  • Such vectors make it possible in a particularly simple manner to modify the system of the sucrose transport in a plant in a targeted and comprehensive manner.
  • the invention also relates to host cells which contain one of the nucleic acid molecules according to the invention or one of the vectors according to the invention stably integrated or transiently or are transformed with it and are preferably capable of SUT4 and optionally SUT1 and / or To express SUT2.
  • the invention further relates to host cells which are derived from a host cell transformed with the nucleic acid molecules according to the invention or the vectors according to the invention.
  • the invention thus relates to host cells which contain the nucleic acid molecules or vectors according to the invention, a host cell being understood to mean an organism which is capable of taking up recombinant nucleic acid molecules in vitro and, if appropriate, of synthesizing the proteins coded by the nucleic acid molecules of the invention.
  • the invention relates to microorganisms which contain the vectors, derivatives or parts of the vectors according to the invention which enable them to synthesize proteins with sucrose transport activity.
  • the host cell according to the invention can also be characterized in that the introduced nucleic acid molecule according to the invention is either heterologous with respect to the transformed cell, that is to say naturally does not occur in this cell, or is located at a different location or a different number of copies in the genome than the corresponding naturally occurring sequence.
  • this host cell is therefore a procaryotic cell, preferably a gram-negative procaryotic cell, particularly preferably an enterobacterial cell.
  • the transformation of procaryotic cells with exogenous nucleic acid sequences is familiar to a person skilled in the field of molecular biology.
  • the cell according to the invention can also be a eucaryotic cell, such as a plant cell, a fungal cell, for example yeast or an animal cell. Methods for the transformation or transfection of eucaryotic cells with exogenous nucleic acid sequences are familiar to the person skilled in the field of molecular biology.
  • the invention also relates to cell cultures or callus tissues which have at least one of the host cells according to the invention, the cell culture or callus according to the invention being in particular able to produce a protein with sucrose transport activity.
  • the nucleotide sequence used according to the invention is linked in the vector to a nucleic acid molecule which encodes a functional signal sequence for forwarding the protein to different cell compartments or the plasma membrane.
  • This modification can consist, for example, of an addition of an N-terminal signal sequence of a higher plant, but also any other modification which leads to the fusion of a signal sequence to the encoded protein is the subject of the invention.
  • nucleic acid molecules according to the invention in procaryotic cells, for example in Escherichia coli, or in eucaryotic cells, for example in yeast, is interesting in that z. B. a more precise characterization tion of the activities of the proteins encoded by these molecules.
  • Another embodiment according to the invention are, preferably purified and isolated peptides or proteins, coded by the nucleotide sequences according to the invention, in particular with the amino acid sequences of SEQ ID Nos. 5 to 8, 23 to 26, 28 or 30, preferably with the activity of a sucrose transporter, in particular a sucrose transporter with low affinity for sucrose and a high transport capacity for sucrose, or with the activity of a sensor or regulator of sucrose transport, and methods for their production, wherein a host cell according to the invention is cultivated under conditions which allow the synthesis of the protein and then that Protein is isolated from the cultured cells and / or the culture medium.
  • the invention further relates to the monoclonal or polyclonal antibodies which react specifically with these proteins.
  • nucleic acid molecules according to the invention it is possible, using genetic engineering methods, to modify the sucrose transport in the tissues of any plant, as is done by conventional, e.g. B. breeding measures in plants was not possible, and to change it so that there is a targeted change in sucrose concentration in certain tissues of a plant.
  • increasing the activity of the proteins according to the invention For example, by overexpressing corresponding nucleic acid molecules, or by providing mutants that are no longer subject to the cell's regulatory mechanisms and / or have different temperature dependencies in relation to their activities, there is the possibility of increasing the yield in correspondingly genetically modified plants.
  • nucleic acid molecules used according to the invention in plant cells in order to increase the activity of the corresponding sucrose transporters, or to express them in cells which do not normally express this protein. Furthermore, it is possible to modify the nucleic acid molecules used according to the invention by methods known to the person skilled in the art in order to obtain proteins according to the invention which are no longer subject to the cell's own regulatory mechanisms or which have changed temperature dependencies or substrate or product specificities.
  • the invention also provides that the synthesized protein can be located in any compartment or in the plasma membrane of the plant cell. In order to achieve localization in a particular compartment or the plasma membrane, the coding region may have to be linked to DNA sequences which ensure the localization in the respective compartment or the plasma membrane.
  • the present invention thus also relates to transgenic plant cells which have been transformed with one or more nucleic acid molecule (s) according to the invention or used according to the invention, and to transgenic plant cells which are derived from such transformed cells.
  • Such cells contain one or more nucleic acid molecules (s) used or according to the invention, these (s) preferably being linked to regulatory DNA elements which ensure transcription in plant cells, in particular with a promoter.
  • the invention also relates to transgenic plant cells whose genome contains at least two stably integrated modified genes from the family of the SUT and / or SUC genes.
  • Such cells can be distinguished from naturally occurring plant cells in that they contain at least one nucleic acid molecule according to the invention or used according to the invention which does not naturally occur in these cells, or in that such a molecule is integrated at a location in the genome of the cell where it is does not occur naturally, ie is in a different genomic environment or in a number other than the natural number of copies.
  • the transgenic plant cells can be regenerated into whole plants using techniques known to those skilled in the art.
  • the plants obtainable by regeneration of the transgenic plant cells according to the invention are also the subject of the present invention.
  • the invention also relates to plants which contain at least one, but preferably a multiplicity, of cells which the cells according to the invention or those used according to the invention Contain vector systems or derivatives or parts thereof, and which are capable of synthesizing proteins due to the inclusion of these vector systems, derivatives or parts of the vector systems, which bring about modified sucrose transport activity, in particular SUT4 activity.
  • the invention thus makes it possible to provide plants of the most diverse types, genera, families, orders and classes which have the abovementioned characteristics.
  • the transgenic plants can in principle be plants of any plant species, ie both monocotyledonous and dicotyledonous plants, such as Graminae, Pinidae, Magnoliidae, Ranunculidae, Caryophyllidae, Rosidae, Asteridae, Aridae, Liliidae, Arecidae and Commelinidae as well as Gymnospermae, algae, mosses, ferns or also Calli, plant cell cultures etc., as well as parts, organs, tissues, harvesting or propagation materials thereof. It is preferably useful plants, in particular starch-synthesizing or starch-storing plants, such as. B.
  • the invention also relates to other plants such as tomato, arabidopsis, pea, rapeseed, sunflower, tobacco, rye, oats, cassava, lettuce, spinach, wine, apple, coffee, tea, banana, coconut, palm trees, beans, pine, poplar, Eucalyptus etc.
  • the invention also relates to propagation material and harvest products of the plants according to the invention, for example flowers, fruits, seeds, bulbs, roots, leaves, rhizomes, seedlings, cuttings, etc.
  • any promoter active in plants for example a promoter which constitutively expresses, or only in a certain tissue, at a certain time in plant development or at a time, is suitable external influences determined point in time.
  • the promoter can be homologous or heterologous to the plant. Promoters that can be used are e.g. B.
  • the promoter of the 35S RNA of the Cauliflower Mosaic Virus (CaMV) and the ubiquitin promoter from maize for a constitutive expression particularly preferably the patatin gene promoter B33 (Rocha-Sosa et al., Loc. Cit.)
  • the patatin gene promoter B33 (Rocha-Sosa et al., Loc. Cit.)
  • a promoter that ensures expression only in photosynthetically active tissues e.g. B. the ST-LS1 Pro otor (Stockhaus et al., Proc. Natl. Acad. Sci. USA (1987) 84, 7943-7947, Stockhaus et al., EMBO J.
  • HMG promoter from wheat
  • USP promoter the phaseolin promoter or promoters of zein genes from maize.
  • a termination sequence can also be present in the vector, which serves to correctly terminate the transcription and to add a poly-A tail to the transcript in order to stabilize it.
  • Such elements are described in the literature (Gielen et al., EMBO J. (1989) 8, 23-29) and are interchangeable. Further promoters are described above.
  • a large number of cloning vectors are available for the introduction of foreign genes into higher plants, which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells.
  • examples of such vectors are pBR322, pUC series, Ml3mp series, pACYCl ⁇ l etc.
  • the desired sequence can be introduced into the vector at a suitable restriction site.
  • the plasmid obtained is used for the transformation of z.
  • Transformed E. coli cells are grown in a suitable medium, then harvested and lysed.
  • the plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained.
  • the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences.
  • Each plasmid DNA sequence can be cloned into the same or different plasmids.
  • a variety of techniques are available for introducing DNA into a plant host cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the biolistic method and others Possibilities.
  • Simple plasmids such as e.g. B. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, a selectable marker should be present.
  • SUT1, SUT2 and / or SUT4 coding nucleotide sequences into the plant cell, additional DNA sequences may be required.
  • z. B. for the transformation of the plant cell uses the Ti or Ri plasmid, at least the right border sequence, but often the right and left border sequence of the Ti and -Ri plasmid T-DNA as the flank region must be linked to the genes to be introduced , If agrobacteria are used for the transformation, the DNA to be introduced must be cloned into special plasmids, either in an intermediate vector or in a binary vector.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria by means of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria.
  • the intermediate vector can be transferred to Agrobacterium tumefaciens using a helper plasmid.
  • Binary vectors can replicate in both E. coli and agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border regions. They can be transformed directly into the agrobacteria (Holsters et al., Mol. Gen.
  • the agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. Additional T-DNA may be present.
  • the agrobacterium transformed in this way is used to transform plant cells.
  • the use of T-DNA for the transformation of plant cells is described in EP-A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters. BV, Alblasserdarn (1985), Chapter V, Fraley et al. , Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. (1985) 4, 277-287.
  • plant explants can be co-cultivated with Agrobacterium tu efaciens or Agrobacterium rhizogenes.
  • Agrobacterium tu efaciens or Agrobacterium rhizogenes From the infected plant material z. B. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells can then be regenerated again in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells, whole plants.
  • the plants thus obtained can then be examined for the presence of the introduced DNA.
  • Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known (Willmitzer, L., 1993 Transgenic plants, In: Biotechnology, A Multi-Volume Comprehensive Treatise (HJ Rehm, G. Reed, A.
  • the invention also relates to identification and screening methods for modulators of sucrose metabolism, preferably potential pesticides and herbicides, SUT4 and / or SUT2-expressing cells or tissues, in particular host cells or plants according to the invention, for example yeast or plant cells according to the invention in contact with the one to be examined brought potential modulator, for example pesticide or herbicide and the effect, in particular the inhibitory effect of the potential modulator, for example pesticide or herbicide, on the activity of SUTl, SUT2 and / or SUT4 is quantitatively or qualitatively demonstrated.
  • SUT2 and / or SUT4 can also be used to develop systems that enable improved pesticide mobilization.
  • Inhibitors can be identified by screening chemical libraries for substances which specifically block the growth of yeasts which express low-affinity sucrose transporters such as SUT4, or the combinations of other sucrose transporters, for example SUT4 and SUT2 or SUT2 and SUT1 or SUT4 and SUT1 , These inhibitors could be used as herbicides or as precursors for new herbicides. On the basis of the tests, potential pesticides can also be identified, which are mobilized in the plant via these transporters and can thus better reach their destination. ',
  • the invention also includes influencing ki eraplasty, ie influencing the activity of transporters in the plant by using mixed oligonucleotides, thereby increasing the activity the sucrose transporter is either increased, decreased or its biochemical properties are changed.
  • influencing ki eraplasty ie influencing the activity of transporters in the plant by using mixed oligonucleotides, thereby increasing the activity the sucrose transporter is either increased, decreased or its biochemical properties are changed.
  • the invention therefore also relates to the use of SUT2-coding nucleotide sequences or of SUT2 for the identification of modulators, in particular inducers, activators or inhibitors of sucrose transport, in particular the sensor system and / or regulation of sucrose transport in a plant, the activity of the protein encoded by the SUT2-encoding nucleotide sequences in the presence and absence of a potential modulator. It can also be provided that not the activity of SUT2, but a sucrose transporter regulated by SUT2, e.g. Evidence of SUTl or SUT4.
  • the invention also relates to the use of SUTl-coding nucleotide sequences, in particular SEQ ID No. 22 and / or of SUT4-coding nucleotide sequences and / or of SUTl and / or SUT4 for the identification of modulators of sucrose transport activities in a plant, in particular an inhibitor the low-affinity, high-capacity loading of the phloem with sucrose, the activity of a protein encoded by the SUT4 nucleotide sequences in the presence of the absence and absence of the potential modulator is demonstrated.
  • SUTl-coding nucleotide sequences in particular SEQ ID No. 22 and / or of SUT4-coding nucleotide sequences and / or of SUTl and / or SUT4 for the identification of modulators of sucrose transport activities in a plant, in particular an inhibitor the low-affinity, high-capacity loading of the phloem with sucrose, the activity of a protein encoded by the SUT
  • the invention also relates to the use of the SUT1, SUT2 and SUT4 nucleotide sequences according to the invention for the identification of homologous genes in other plants, e.g. from cDNA or genomic banks.
  • the invention also relates to the use of the genes and the surrounding regions as molecular markers for crossing programs.
  • SUT2 and SUT4 loci are located in regions of QTL loci for high carbohydrate content and high yield in potato tubers. Therefore, both are suitable for crossing suitable chromosome fragments in breeding programs from wild forms or high-performance varieties and thus producing plants with improved yields.
  • SEQ ID No. 1 the coding DNA sequence of the SUT4 gene from Arabidopsis thaliana.
  • SEQ ID No. 2 the coding DNA sequence of the SUT4 gene from Lycopersicon esculentum.
  • SEQ ID No. 3 the coding DNA sequence of the SUT2 gene from Arabidopsis thaliana.
  • SEQ ID No. 4 the coding DNA sequence of the SUT2 gene from Lycopersicon esculentum.
  • SEQ ID No. 5 the amino acid sequence of SUT4 from Arabidopsis thaliana.
  • SEQ ID No. 6 the amino acid sequence of SUT4 from Lycopersicon esculentum.
  • SEQ ID No. 7 the amino acid sequence of SUT2 from Arabidopsis thaliana.
  • SEQ ID No. 8 the amino acid sequence of SUT2 from Lycopersicon esculentum.
  • SEQ ID No. 9 a T-DNA specific primer.
  • SEQ ID No. 10 a SUT4-specific primer.
  • SEQ ID No. 11 a SUT4-specific primer.
  • SEQ ID No. 12 to 15 represent further SUT4 primers.
  • SEQ ID Nos. 16 and 17 represent the amino acid sequence of sections of LeSUT4.
  • SEQ ID No. 18 to 21 represent cloning pri er.
  • SEQ ID No. 22 the coding DNA sequence of the SUT1 gene from Solanum tuberosum.
  • SEQ ID No. 23 the amino acid sequence of SUTl from Solanum tuberosum.
  • SEQ ID No. 24 the DNA sequence of SEQ ID No. 3 coding for the N-terminal region of SUT2 with nucleotides 1 to 239.
  • SEQ ID No. 25 the DNA sequence of SEQ ID No. 22 coding for the N-terminal region of SUT1 with nucleotides 1 to 149.
  • SEQ ID No. 26 the DNA sequence from SEQ ID No. 3 coding for the central loop with nucleotides 843 to 1130.
  • SEQ ID No. 27 the coding DNA sequence of the SUT4 gene from Solanum tuberosum.
  • SEQ ID No. 28 the amino acid sequence of SUT4 from Solanum tuberosum.
  • SEQ ID No. 29 the coding DNA sequence of the SUT2 gene from Solanum tuberosum.
  • SEQ ID No. 30 the amino acid sequence of SUT2 from Solanum tuberosum.
  • FIGS 1 to 4 show schematically the structure of gene constructs used according to the invention.
  • FIGS. 5 and 6 show graphic representations of the protein activities of SUT 4.
  • FIG. 7 shows chimeras SUT2 and SUT1 gene constructs according to the invention.
  • Genomic sequence AtSUT4 AC000132
  • a cDNA was amplified from an Arabidopsis seedling bank using PCR (Minet et al., Plant J. (1992) 2, 417-422). Primers based on the genomic sequence (5 '-Gactctgcagcgagaaatggctacttccg SEQ ID No. 12, 5' -taacctgcaggagaatctcatgggagagg SEQ ID No. 13) were designed. The primers each contain a PstI restriction site (underlined) and are designed so that the entire coding sequence of AtSUT4 is amplified.
  • AtSUT4 was subcloned into the PstI site of pDR196.
  • the AtSUT4 cDNA in pDR196 was sequenced in both directions. Ecotype differences between the SUT4 gene in Columbia and Landsberg erecta were verified by sequencing AtSUT4 from Landsberg erecta.
  • a tomato (Lycopersicon esculentum cv. UC82b) cDNA library (flowers) was treated with a 300 bp Eco-RIBglll fragment of the genomic tobacco clone NtSUT3 (Lemoine et al., FEBS Lett. (1999) 454, 325-330). through the reduced stringency.
  • Three independent clones different from LeSUT1 were isolated and designated LeSUT4. With primers of the LeSUT4 sequence, the orthologous sequence was isolated from potato by RT-PCR and designated StSUT4.
  • StSUT4 was cloned as a Pstl / Notl fragment in pBC SK- (Stratagene) and subcloned as an XhoI / SacII fragment into the yeast expression vector pDR195, which contains a URA3 marker, PMAl promoter and ADHl terminator (Rentsch et al., FEBS Lett. (1995) 370, 264-268).
  • Genomic sequence AtSUT2 AC004138
  • Primers based on the genomic sequence (5'-TACGAGAATTCGATCTGTGTGTTGAGGACG, SEQ ID No. 20, 5'-AGAGGCTCGAGTGGTCAAAAAGAATCG, SEQ ID No. 21) were designed.
  • the primers contain an EcoRI or an Xhol restriction site (underlined) and are designed to amplify the entire AtSUT2 coding sequence.
  • a product with the expected size of 1785 bp was cut with EcoRI and Xhol and ligated directionally into the vector pDR19 ⁇ .
  • the yeast strain SUSY7 / ura3 is a modified version of SUSY7 (Riesmeier et al., EMBO J. (1992) 11, 4705-4713), which contains a deletion of part of the URA3 gene and thereby enables selection for uracil auxotrophy.
  • sucrose medium media were used which contain 1.7 g / 1 yeast nitrogen base without amino acids (Difco), 2% sucrose, 20 mg / 1 tryprophan and 1.5% agarose, pH 5.0 included.
  • At-SUT4 and StSUT4 proved to be functional sucrose transporters.
  • the time course of the uptake of 14 C-sucrose from the yeast expressing AtSUT4 or StSUT4 is shown in FIG. 5A. There is a significant difference to the vector controls.
  • FIG. 5C shows the stimulation of the uptake of 14 C-sucrose via SUT4 by glucose and the inhibition by an electron transport inhibitor (antimycin A and the protonophores CCCP.
  • FIG. 6 shows the pH optimum of SUT4-mediated sucrose transport.
  • T-DNA mutagenized Arabidopsis plants were obtained from Dupont Co. and from Arabidopsis Biologi- cal Resource Center (ABRC), Ohio State University. The plants were examined in groups of 100. The plants were grown in sterile culture and genomic DNA isolated. The DNA from the 140 groups of 100 plants each was combined into 14 supergroups (superpool) and according to the method of Krysan et al. (Proc. Natl. Acad. Sci. USA (1996) 93, 8145-8150), using gene-specific and T-DNA-specific primers. A PCR was carried out using the Superpool DNA as a template, a T-DNA-specific primer (LB, left border region SEQ ID No.
  • PCR products were separated by agarose gel electrophoresis and transferred to a charged nylon membrane.
  • the membrane was hybridized with a PCR product of 2.46 kb in length, prepared from WS (Wassilewskija) genomic DNA as a template and the primers AtSUT4r2 (see above) and AtSUT4f2 (ATGGCTACTTCCGATCAAGATCGCCGTC SEQ ID No. 11). This probe was labeled with 32 P-CTP.
  • a super pool was identified by hybridizing the labeled probe with the blot.
  • DNA from the pools of 100 plants that form the superpool were then screened in the same way: PCR was carried out using DNA from pools of 100 as a template and AtSUT4r2 and LB as a primer, DNA blot hybridization was carried out using the AtSUT4 genomic probe ( 2.46 kb) carried out for the detection of amplified products.
  • Preimmune serum was purified by the same method, except that Protein A Sepharose (BioRad) was used instead of peptide affinity chromatography.
  • the slides were incubated for 30 seconds in acetone, rehydrated over an ethanol series and blocked for 1 hour with 2% BSA in PBS (100 mM sodium phosphate, pH 7.5, 100 mM NaCl). After overnight incubation with affinity-purified antibodies to LeSUT4, the slides were washed twice in PBS-T (PBS with 0.1% Tween) and once with PBS, followed by incubation with anti-rabbit conjugate IgG-FITC (fluorescinisothio-cyanate) for one hour ) washed. After three washing steps with PBS-T, PBS and distilled water, pictures were taken with a fluorescence phase microscope (Zeiss, Axiophot) and an excitation light of 450-490 nm.
  • PBS-T PBS with 0.1% Tween
  • IgG-FITC immunofluorescinisothio-cyanate
  • AtSUT2 cDNA was amplified by PCR - the product was 1,785 bp long in accordance with the coding range according to the invention from ATG (position 1) to TGA (position 1785).
  • the fragment was cloned in a sense orientation into a 35S promoter Expression cassette (pBinAr35S), which was isolated as an EcoRI / HindIII fragment from pBinAr (Höfgen and Willmitzer, Plant Sc. (1990) 66, 221-230).
  • This construct was cut with HindIII and EcoRI and cloned into the HindIII / EcoRI-cut pGTPV bar (Becker et al., Loc. Plant Mol. Biol. (1992) 20, 1195-1197). Plants were transformed.
  • AtSUT2 antisense construct ( ⁇ AtSUT2 35s )
  • AtSUT2 cDNA (ATTS5034EST accession number) was cut with SacI and BamHI and cloned into the pBinAr35S expression cassette in antisense orientation. This construct was cut with HindIII and EcoRI and cloned into the HindIII / EcoRI-cut pGPTV bar (Becker et al., Op. Cit.). Plants were transformed.
  • AtSUT4 overexpression construct (oAtSUT4 AtS uc2)
  • AtSUT4 cDNA was amplified.
  • the 1,533 bp fragment begins by means of PCR at position 1 of the AtSUT4cDNA sequence according to the invention and ends at TAG position 1533.
  • the SUC2 promoter was separated from Arabidopsis (Columbia ecotype) genomic DNA using the following primers: (reverse 5'-ATGGCTGACCAGATTTGAC; SEQ ID No. 18 and forward 5'- GTTTCATATTAATTTCAC; SEQ ID No. 19).
  • the 1.533 kb fragment was cloned in the sense orientation behind the At-SUC2 promoter (X79702). This construct was cut with Hindlll and EcoRI and cloned into the Hindlll / EcoRI-cut pGPTV-bar (loading cker et al., loc. cit.). Plants were transformed.
  • LeSUT4 antisense construct ( ⁇ LeSUT4 35s )
  • the LeSUT4 cDNA was cut with BamHI to give a 1.3 kb fragment which was smoothed and cloned into the Smal site of pBinAR (Bevan, Nucleic Acids Research (1983) 12, 8711-8721).
  • FIGS. 1 to 4 represent the aforementioned constructions.
  • AtSUT2 The open reading frame of AtSUT2 was isolated from Arabibopsis thaliana (Columbia Ecotyp) leaves by RT-PCR and cloned into the yeast expression sector pDR196 (Barker et al., (2000) Plant Cell 12: 1153-1164).
  • the open reading frame of StSUT1 was amplified by the StSUTl cDNA in pDRl95 (Riesmeier et al., (1993) op. Cit.), Using primers with the restriction sites for Smal and Xhol.
  • the open reading frame was ligated into the yeast expression vector pDRl96.
  • StSUTl / AtSUT2-N has the nucleotides 1 to 239 from SEQ ID No. 3 fused to the nucleotides 150 to 1548 from StSUTl, shown in SEQ ID No. 22, the construct being a ' nucleotide due to cloning conditions d exchange from t to c.
  • the fusion region of the construct is shown in sequence below, the small letters being the sequences of SUT2 and the large letters being the sequences of SUT1 (upper line: without exchange, lower line: with exchange):
  • AtSUT2 / StSUTl-N has nucleodides 1 to 149 of StSUT1, shown in SEQ ID No. 22, fused to nucleotides 240 to 1785 of AtSUT2 in SEQ ID No. 3. Due to technical cloning conditions, the construct has 3 nucleotide changes compared to the wild-type sequence.
  • the fusion area is shown below, in the upper line the theoretically obtainable construct and in the lower line the fusion area actually produced.
  • the upper case letters refer to sequences from SUT1 and the lower case letters to sequences from SUT2:
  • chimeric constructs were produced in which the central cytoplasmic area, in particular loop, of AtSUT2, which is shown in SEQ ID # 26, was exchanged with the smaller cytoplasmic area, in particular loop, of StSUT1 and vice versa. Restriction interfaces were used by means of PCR within conserved areas of the transmembrane regions VI and VII.
  • the N-terminal half, the cytoplasmic loop and the C-terminal half of the open reading frame were amplified by PCR using Pfu polymerase (Stratagene) and cloned into the yeast expression vector by ligating the first three fragments using Sac I and Bcll / Bglll for AtSUT2 with the StSUTl loop and Sacl and BamHI / Bglll for StSUTl with the AtSUT2 loop.
  • the chimeric DNA was then ligated into the yeast expression vector pDR196 using Smal and Xhol. These chimeric constructs are still called AtSUT2 / StSUTl-Loop and StSUTl / AtSUT2-Loop designated and shown in Figure 7.
  • AtSUT2 / StSUTl-Loop has nucleotides 1 to 842 from AtSUT2 (SEQ ID No. 3), 750 to 893 from StSUT1 (SEQ ID No. 22) and nucleotides 11.31 to 1785 from AtSUT2 (SEQ ID No. 3).
  • the upper and lower case representation used in the following corresponds to the aforementioned use.
  • the upper line represents the sequence of the theoretically obtainable construct and the respective lower line represents the sequence of the construct actually achieved on the basis of cloning conditions.
  • StSUTl / AtSUT2-Loop has nucleotides 1 to 749 from StSUTl (SEQ ID No. 22), nucleotides 843 to 1130 from AtSUT2 (SEQ ID No. 3) and nucleotides 894 to 1548 from StSUTl (SEQ ID No. 22) on .
  • the yeast strain SEY6210 (Banakaitis (Proc. Natl. Acad. Sei. USA (1986) 83, 9075-9070), which had the corresponding cDNAs in the expression vector pDR196, was used for sucrose uptake tests.
  • the uptake of 14-C-sucrose was carried out as described ( Weise et al., (2000) Plant Cell 12: 1345-1355)
  • Expression analysis of the proteins in yeast revealed comparable amounts for all proteins examined.
  • sucrose uptake by AtSUT2 was pH-dependent, the highest uptake rates being measured at a pH of 4.0.
  • the sucrose uptake dropped sharply at alkaline pH values and at a pH value of 6 no more sucrose uptake could be measured.
  • the sucrose uptake (1 mM sucrose) was competitively measured with other sugars and sugar alcohols.
  • sucrose maltose, isomaltulose, glucomannitol, glucosorbitol, raffinose, galactose, lactose, mannitol, sorbitol, glucose
  • sucrose competed and to a lesser extent maltose significantly with 1 C-sucrose.
  • Sucrose transport by AtSUT2 could be inhibited by CCCP and by the mitochondrial ATP formation inhibitor, Antimycin A.
  • K M values for sucrose of the StSUT1 and AtSUT2 sucrose transporters and chimeric proteins in which the N-terminal regions or central cytoplasmic loops were exchanged between the two transporters The values are given as the mean ⁇ standard error determined from at least three different measurements. Different letters indicate significant differences (p ⁇ 0.05).
  • the chimeric protein encoded by the chimeric construct AtSUT2 / StSUTl-N shows a significantly lower K M value for sucrose of 3.4 + 1.6 mM

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Abstract

L'invention concerne une molécule d'acide nucléique codant pour un saccharide, en particulier pour un transporteur de saccharose, des vecteurs et des cellules hôtes contenant ces molécules d'acide nucléique, ainsi que les cellules végétales et les plantes transformées par les molécules d'acide nucléique et les vecteurs précités. En outre, l'invention concerne des procédés de modification du transport de saccharide, en particulier du saccharose, dans des plantes.
EP01905823A 2000-03-24 2001-02-26 Procede de modification genetique d'une plante Withdrawn EP1276882A2 (fr)

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DE10014672 2000-03-24
DE10014672 2000-03-24
DE10050233 2000-10-11
DE10050233A DE10050233A1 (de) 2000-03-24 2000-10-11 Verfahren zur genetischen Modifizierung einer Pflanze
PCT/EP2001/002148 WO2001073086A2 (fr) 2000-03-24 2001-02-26 Procede de modification genetique d'une plante

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WO2011062748A1 (fr) * 2009-11-23 2011-05-26 E.I. Du Pont De Nemours And Company Gènes des transporteurs du saccharose pour augmenter les lipides des graines végétales
WO2012068445A2 (fr) * 2010-11-18 2012-05-24 University Of Georgia Research Foundation, Inc. Modification de la répartition du saccharose dans les plantes
CN105189759A (zh) * 2013-03-13 2015-12-23 华盛顿卡内基研究所 调节植物种子和蜜腺内含物的方法
CN116675751B (zh) * 2023-06-08 2024-01-26 山东农业大学 SWEET1g蛋白及其编码基因在抗马铃薯病毒中的应用

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DE4220759A1 (de) * 1992-06-24 1994-01-05 Inst Genbiologische Forschung DNA-Sequenzen für Oligosaccharid-Transporter, Plasmide, Bakterien und Pflanzen enthaltend einen Transporter sowie Verfahren zur Herstellung und Transformation von Hefestämmen zur Identifikation des Transporteers
EP1070130A2 (fr) * 1998-04-09 2001-01-24 E.I. Du Pont De Nemours And Company Transporteurs de sucrose d'origine vegetale

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WO2001073086A2 (fr) 2001-10-04
JP2004500827A (ja) 2004-01-15
BR0109492A (pt) 2003-04-29
CA2402098A1 (fr) 2001-10-04

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