EP1183379A2 - Systeme promoteur, sa production et son utilisation - Google Patents

Systeme promoteur, sa production et son utilisation

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Publication number
EP1183379A2
EP1183379A2 EP00949099A EP00949099A EP1183379A2 EP 1183379 A2 EP1183379 A2 EP 1183379A2 EP 00949099 A EP00949099 A EP 00949099A EP 00949099 A EP00949099 A EP 00949099A EP 1183379 A2 EP1183379 A2 EP 1183379A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
invertase
plant
promoter
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00949099A
Other languages
German (de)
English (en)
Inventor
Thomas Dr. Roitsch
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.)
Freistaat Bayernvertreten Durch Die Julius-Maximil
Original Assignee
Julius Maximilians Universitaet Wuerzburg
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Julius Maximilians Universitaet Wuerzburg filed Critical Julius Maximilians Universitaet Wuerzburg
Publication of EP1183379A2 publication Critical patent/EP1183379A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds

Definitions

  • the present invention relates generally to gene expression and its regulation in plants. More particularly, the present invention relates to nucleic acid sequences coding for a promoter, expression systems comprising them, nucleic acid constructs, vectors and cells, plants, seeds obtainable from plants and methods for producing male-sterile plants. More particularly, the invention relates to DNA promoter sequences and expression cassettes that can be introduced into plants to regulate the transcription of an adjacent coding sequence in time and space within the plants.
  • a promoter is a DNA sequence which influences or determines the expression site and the expression amount of a gene and which provides the sites for the binding of the RNA polymerase.
  • the position of a promoter is fixed in the genome of an organism relative to the start of transcription.
  • RNA polymerase is an enzyme that can bind to the promoter and transcribe a gene that is under the control of this promoter. This creates the messenger RNA (mRNA), which in turn is used to synthesize the protein.
  • Promoters have been studied in various organisms. For certain species, conserved DNA areas (so-called consensus sequences) could be found within promoters that are associated with different genes. These areas are believed to be involved in the role that the promoter plays in the transcription process.
  • the initiation of the transcription process in plants includes one Interaction of the promoter with RNA polymerase II. Consensus sequences were found within plant promoters above the 5 'end of the transcription start point. One of these sequences is about 7 base pairs long and is located about 20-30 base pairs above the transcription start point. This sequence is known as a so-called TATA box and is believed to play a role in RNA polymerase binding. Another sequence, approximately 9 base pairs in length, is found approximately 70-90 base pairs above the transcription start point.
  • CAAT-box This sequence is called CAAT-box and is believed to play a role in regulating the level of transcription.
  • Other regions above the start of transcription have been identified that affect the frequency of initiation of transcription in eukaryotes. These areas of DNA called enhancers affect the activity of promoters in their neighborhood. However, these sequences are by definition not promoters since their position need not be fixed.
  • the coding sequence of this gene must be placed under the control of a promoter and introduced into the plant.
  • the foreign DNA is mostly placed in the Ti plasmid of Agrobacterium tumefaciens, and this is then used to transform the plants.
  • a second frequently used method is the direct transformation of DNA e.g. with the help of the so-called "particle gun”.
  • promoters or plant virus promoters isolated from bacteria have been used to date, which lead to the expression of the foreign gene in the plants. For certain applications, these promoters have the disadvantage that they are foreign to the species and are therefore not subject to the control mechanisms within the plants.
  • the object of the present invention is to provide a promoter which is suitable for controlling the expression of nucleic acids in plants or plant cells.
  • a partial aspect of the task is to provide promoters which have a high expression with simultaneous tissue specificity.
  • the object of the invention is to provide a method for producing male-sterile plants.
  • the object is achieved in one aspect by a nucleic acid sequence coding for a promoter, the promoter being both tapetum-specific and pollen-specific.
  • the object is achieved by a nucleic acid sequence coding for a promoter, the nucleic acid sequence comprising a region of at least about 900 nucleotides upstream of the TATA box of the sequence shown in SEQ ID No. 1.
  • the nucleic acid sequence comprises a region of at least about 1000 nucleotides upstream of the TATA box of the sequence shown in SEQ ID No. 1.
  • the nucleic acid sequence comprises a region of at least approximately 1500 nucleotides upstream of the TATA box of the sequence shown in SEQ ID No. 1.
  • nucleic acid sequence the in SEQ ID NO. 1 sequence shown includes.
  • the object is also achieved by a nucleic acid sequence coding for a promoter, the nucleic acid sequence comprising the sequence shown in SEQ ID No.2.
  • the object is achieved by a nucleic acid sequence coding for a promoter, the nucleic acid sequence comprising the sequence shown in SEQ ID No.3.
  • the object is achieved by an expression system comprising at least one of the nucleic acids according to the invention.
  • the expression system further comprises at least one terminator and / or one linker.
  • nucleic acid construct comprising a nucleic acid sequence according to the invention and at least part of an expressible nucleic acid sequence.
  • part of the expressible nucleic acid sequence or the complete expressible sequence is connected in the sense direction to one of the nucleic acid sequences according to the invention.
  • the expressible nucleic acid codes for an invertase.
  • the part of the nucleic acid sequence of an invertase or the complete sequence of an invertase is linked with that of one of the nucleic acid sequences according to the invention in the antisense direction.
  • the invertase is one which is present in a structure which is selected from the group comprising anthers, tapetum, pollen precursor cells and pollen.
  • the invertase comes from the organism into which or in whose cells the nucleic acid construct is to be introduced, and in particular comes from the group of plants to which the species into which the nucleic acid construct is to be introduced belongs. In a still further embodiment it is provided that the organism is selected from the group comprising useful plants, ornamental plants and medicinal plants.
  • the object is achieved by a vector comprising one of the nucleic acid sequences according to the invention and / or an expression system 9 according to the invention and / or a nucleic acid construct according to the invention.
  • the object is achieved by a cell, in particular a plant cell, comprising a nucleic acid according to the invention and / or an expression system according to the invention and / or a nucleic acid construct according to the invention and / or a vector according to the invention.
  • the cell comprises a nucleic acid sequence according to the invention which is a promoter and a nucleic acid which codes for an inhibitor of an invertase, the promoter controlling the expression of the inhibitor.
  • the cell is selected from the group comprising pollen cells, pollen precursor cells and cells of the tapetum.
  • the cell is a locked pollen cell.
  • the object is achieved by a plant comprising a cell according to the invention.
  • the plant is selected from the group comprising useful plants, ornamental plants and medicinal plants, preferably selected from the group comprising rice, corn, potatoes, tomatoes and rapeseed.
  • the plant is a male sterile plant and has at least one further change in its genotype, in particular a change due to genetic engineering.
  • the object is achieved by a seed that can be obtained from a plant according to the invention.
  • the object is achieved by a hybrid seed which can be obtained by crossing a male sterile plant according to the invention with another male fertile plant and is obtained from the branch generation of the hybrid seeds which has arisen in this way.
  • the object is achieved by a method for producing male sterile plants, a nucleic acid construct according to the invention being introduced into a cell, in particular into a plant cell, and a plant being produced from this cell.
  • the plant is selected from the group comprising useful plants, ornamental plants and medicinal plants, preferably selected from the group comprising rice, corn, potatoes, tomatoes and rapeseed.
  • the object is achieved by using a nucleic acid construct according to the invention for the production of male-sterile plants.
  • the object is achieved by using a nucleic acid sequence according to the invention for expressing a nucleic acid sequence.
  • the object is achieved by a restorer plant, wherein in a cell, preferably in the majority of its cells, it comprises a nucleic acid according to the invention as a promoter and a nucleic acid coding for a further invertase, which is controlled by this promoter, wherein the other invertase is different from the cell's own invertase.
  • a restorer plant which can preferably be one as described above, it being provided that it has a nucleic acid according to the invention as a promoter and in a cell, preferably in the majority of its cells comprises a nucleic acid coding for a sucrose transport system which is controlled by this promoter.
  • a cell in a cell, preferably in the majority of its cells, it further comprises a nucleic acid according to the invention as a promoter and a nucleic acid coding for sucrose synthase and / or cytoplasmic expressed invertase, the expression of which is controlled by the promoter.
  • the object is achieved by a plant which is characterized in that it comprises an inventive nucleic acid construct in at least one cell, preferably in the majority of its cells, and the cell (s) furthermore an inventive nucleic acid sequence as promoter and one for comprises a further invertase-encoding nucleic acid which is controlled by this promoter, the further invertase being different from the cell's own invertase.
  • the object is achieved by a plant which is characterized in that it comprises an inventive nucleic acid construct in at least one cell, preferably in the majority of its cells, and the cell (s) furthermore an inventive nucleic acid sequence as promoter and one for comprises a nucleic acid encoding a sucrose transport system which is controlled by this promoter.
  • the plant also includes the features of the plant according to the seventeenth aspect of the present invention.
  • a further embodiment provides that the plant comprises a nucleic acid construct according to the invention in at least one cell, preferably in the majority of its cells, and the cell (s) furthermore a nucleic acid sequence according to the invention as a promoter and an invertase expressed for sucrose synthase and / or cytoplasmic encoding nucleic acid, the expression of which is controlled by the promoter.
  • a still further embodiment provides that the further invertase, which is different from the cell's own invertase, is selected from the group of invertases, the invertase (s) from Saccharomyces cerevisiae and invertase (s) from Zymomonas mobilis.
  • the sucrose synthase is of heterologous or homologous origin.
  • the invertase expressed cytoplasmic is of homologous or heterologous origin.
  • the cytoplasmic expressed invertase is of heterologous origin and is preferably selected from the group of invertases which include invertase (s) from Saccharomyces cerevisiae and invertase (s) from Zymomonas mobilis.
  • the object is achieved by a seed that can be obtained from a plant according to the invention.
  • the object is achieved by using the seeds according to the invention for the in vitro embryogenesis of haploid or diploid or double-diploid plants.
  • the object is achieved by a fruit, in particular seedless fruit, which can be obtained from one of the plants according to the invention.
  • the object is achieved by a fruit which can be obtained from one of the plants according to the invention, in particular from a restorer plant according to the invention and its hybrid products according to the invention.
  • the object is achieved by a method for cloning promoters which are functionally homologous to one of the promoters according to one of the preceding claims, the method being characterized by the following steps: a) Cloning of anther-specific invertase cDNA by RT-PCR on mRNA from anthers, in particular using the oligonucleotides OIN3 and OIN4 b) Cloning of the corresponding promoters
  • the present invention is based on the surprising finding that promoters exist which are suitable for the expression of nucleic acids in plant cells and thereby show a double tissue specificity.
  • the nucleic acids disclosed herein, which code for a promoter, hereinafter also referred to as promoters for short, have at least double specificity: they lead to the expression of nucleic acid under their control in the tapetum and in the pollen.
  • the promoters according to the invention are extremely strong and show a characteristic course over the anther development divided into 12 phases, which allows a time-defined, specific expression pattern to be achieved when using the promoters according to the invention. As a result of this both spatial specificity, i.e.
  • Tissue specificity offers such vectors a great advantage over promoters inducible by an exogenous stimulus, such as temperature or presence of certain compounds. If such promoters are contained in the genome of a plant, there is a spatially and temporally specific expression of the nucleic acid (s) under the control of this promoter.
  • the nucleic acid under the control of the promoter according to the invention can be any form of nucleic acid. Accordingly, it can be coding nucleic acids or structural or functional nucleic acids.
  • Coding nucleic acid is to be understood in particular to mean a nucleic acid which codes for a peptide or a protein.
  • the peptide / protein can be, for example, a structure rotein or a peptide / protein which has an enzymatic activity.
  • Structural nucleic acid is to be understood here in particular as a nucleic acid which leads to the formation of complexes, in particular with other molecules. This can include an rRNA and, in particular, an antisense nucleic acid.
  • Functional nucleic acid is to be understood here in particular to mean a nucleic acid which has a specific effect on a system, in particular a biological system. Such a specific effect can, for example, be to promote or inhibit translation or transcription.
  • An example of a functional nucleic acid is an antisense nucleic acid.
  • the promoters according to the invention allow the spatially and temporally determined expression of nucleic acids, in particular genes in plant cells and plants.
  • This can be homologous or heterologous nucleic acids or genes.
  • homologous genes they are those which come from the genetic background of the plant which contains one of the promoters according to the invention.
  • genes or nucleic acid sequences already present in the cell are placed under the control of the promoters according to the invention, either as a supplement or as a substitute.
  • heterologous genes or nucleic acids these are those which do not come from, or are present in, the genetic background of the plant which contains one of the promoters according to the invention.
  • the invention is further based on the surprising finding that it is possible to produce male-sterile plants in particular using one of the promoters according to the invention.
  • a nucleic acid which encodes at least part of an invertase is placed under the control of one of the promoters according to the invention.
  • the invertase is preferably one which is present in pollen and / or in the tapetum and can come from the respective plant species.
  • the invertase encoded by SEQ ID No 15 is one which has been isolated from the pollen of tobacco.
  • a portion of the nucleic acid encoding this invertase is brought under the control of a promoter according to the invention, so that the expression product of the nucleic acid encoding the invertase is used as an antisense Nucleic acid acts and consequently suppresses the expression of the invertase present in the pollen and tapetum.
  • the antisense nucleic acid is produced by functionally coupling the nucleic acid coding for the invertase or a part thereof in the anti-sense direction to the promoter, optionally separated by an additional nucleic acid sequence, for example in the form of a linker. This is accomplished by reading the non-coding or anti-sense strand through the promoter, thus incorporating the nucleic acid coding for the invertase in an inverted manner.
  • the antisense nucleic acid occurs precisely when the invertase occurring in the said tissues is particularly active and must be suppressed in order to overcome the energetic deficiency state described above and / or shift the disaccharide to monosaccharide ratio. Due to the strength of the promoters according to the invention, the antisense nucleic acid is expressed to such an extent that the intrinsic invertase activity in the pollen and tapetum is effectively suppressed. As a result of this mechanism of sterile pollen and male sterile plant formation disclosed herein, the pollen remains arrested at a defined stage of its development.
  • This special stage is referred to as the mononuclear microspore stage, which is otherwise run through as part of normal development - fertile - pollen.
  • This mechanism of male sterility in plants does not only occur in tobacco or tomatoes. Rather, the promoter can be used in any plant or plant species. In principle, the same also applies to the nucleic acid which is functionally linked in antisense orientation to one of the promoters according to the invention and which codes for an invertase or a part thereof.
  • the corresponding constructs according to the invention are those in the construct to select the invertase sequence used so that it is identical to the sequence of the intrinsic invertase or else has a degree of homology with it which allows an interaction of the sense and anti-sense nucleic acid.
  • Co-suppression is understood to mean the effect that in the event of overexpression of a gene which is already present in a plant, this does not lead to an increased formation of the gene Gene encoded peptide / protein leads, but rather to a reduced formation.
  • the effect thus corresponds to that of the antisense construct described herein, comprising one of the promoters according to the invention and a nucleic acid coding for an invertase and functionally coupled thereto in an antisense orientation.
  • Co-suppression has an effect on the transcription level.
  • plants are also understood here generally and particularly as useful plants, ornamental plants and medicinal plants.
  • plants are understood to mean both monocotyledons and dicotyledons.
  • Monocotyledons and dicotyledons represent groups of plants for the purposes of the present invention.
  • the use of invertases can extend to both invertases from or in the case of monocotyledons and from or in the case of dicotyledons.
  • invertases from mono- and dicotyledons
  • Another preferred group of plants in which the various aspects of the invention may be used are the following plants: rice, corn, tomatoes, potatoes, rapeseed, soybeans and sugar beets.
  • the claimed nucleic acid sequences coding for a promoter or promoter structure comprise the sequence according to SEQ. ID. No.l, SEQ. ID. No. 2, SEQ. ID. No. 3 or part of each.
  • sequence of SEQ. ID. No. 1 obtained from tobacco in a functionally oriented experiment and it could be shown that the sequence as such has a promoter activity. It is also known to those skilled in the art that with such experimental approaches, a longer sequence is often obtained, which may have additions at the 5 'or 3' end which are insignificant for the promoter property.
  • sequence according to SEQ. ID. No. 1 further characterized and it was found that a shortening of the said sequence is possible while maintaining the promoter property.
  • a shortening of the SEQ sequence. ID. No. 1 In the present case, a sequence section or region that extends approx.
  • nucleotides or base pairs / bp upstream, ie in the 5 'direction, from the so-called TATA box has proven sufficient for the presence of the promoter activity.
  • Further embodiments of the promoter extend over a region of approximately 1000 nucleotides or approximately 1500 nucleotides upstream of the TATA box.
  • the promoter according to SEQ. ID. No. 2 is a promoter of SEQ ID. No. 1 originating promoter, compared to that according to SEQ. ID. No. 1 is extended by approximately 1 kb
  • sequence according to SEQ. ID. No. 3 originally comes from the tomato. Similar to the case of the on the SEQ sequence. ID. No.l-based promoter can also be shortened in principle within the scope of the skill of the person skilled in the art or with additional elements, such as e.g. enhancers, are supplemented while maintaining the promoter property.
  • promoters which correspond to the location and time-specific expression pattern of the promoters according to the invention and are thus functionally homologous to them.
  • anther-specific invertase cDNA is cloned to mRNA from anthers by RT (reverse transcriptase) -PCR.
  • RT reverse transcriptase
  • the promoters controlling these are cloned in a manner as described in the examples herein.
  • the various uses of the promoters described herein are not limited to the promoters disclosed herein and characterized by their sequence, but extend to all those promoters that have double tissue specificity described herein.
  • any of the promoters according to the invention can be introduced into an expression system and can be part of an expression system.
  • the expression systems are preferably expression systems suitable for expression in plants. Expression systems, their components (such as linkers, terminators, insertion sequences, markers etc.) and structure are described in the literature, such as, for example, Asubel, FM; Brent, R .; Kingston, RE; et al. (eds.) (1999) Current protocols in molecular biology. John Wiley & Sons, Massachusetts; Sambrook, J .; Fritsch, EF; Maniatis, T .; (1989) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Clark, MS (1997) Plant molecular biology - a laboratory manual.
  • Expression systems of this type can also be expression cassettes, restriction sites in particular being present at a suitable distance from the promoter and other cassette components, for example through so-called linkers, which allow the nucleic acid sequence to be expressed to be cloned in. If the nucleic acid to be expressed is one that codes for a peptide, polypeptide or protein, it should be particularly noted that the cloning takes place in the reading frame.
  • the expression systems according to the invention or the vectors containing them can be transformed into plants or plant cells using techniques known in the art (reference see above) (e.g. Agrobacterium-mediated transformation; direct transformation etc.)
  • the nucleic acid constructs according to the invention comprise at least one of the promoters according to the invention and at least part of an expressible nucleic acid sequence. Depending on the type of the expressible nucleic acid sequence and its positioning relative to the promoter, different fields of application of these nucleic acid constructs arise.
  • An expression product can be an mRNA, which in turn is translated into a peptide or protein.
  • the translation product can either have a direct effect (show in a biological system) or an indirect effect.
  • a direct effect would be, for example, the production of a cytotoxin based on a peptide or the production of a structural protein.
  • An indirect effect would be, for example, the production of an enzyme that catalyzes certain metabolic reactions, which in turn affects the phenotype of the cell or the plant containing it.
  • Another form of an expression product can also be a functional nucleic acid.
  • nucleic acid construct according to the invention in particular that according to SEQ ID No. 8 represents the sterile generation Pollen leads and, if such a construct is present in a plant, more precisely in its genome, leads to male sterile plants.
  • nucleic acid (sequence) to be expressed which is under the control or control of one of the promoters according to the invention, is required, this is typically functionally linked to the promoter in the sense direction or orientation.
  • the sense strand or coding strand is the one that is transcribed (in contrast to the antisense direction or orientation).
  • An example of such a strongly expressing nucleic acid construct is one as described herein for co-suppression and its use for the production of male sterile plants.
  • nucleic acid construct consists of one of the promoters according to the invention and the nucleic acid, or a part thereof, which codes for an inhibitor of an invertase, in particular of the invertase occurring in pollen and / or tapetum.
  • the use of such a construct also represents a possibility of producing male sterility in plants.
  • this measure enables the construct according to the invention, in which a promoter according to the invention is linked to a sequence which codes for an invertase or a part thereof, and which Sequence in the anti-sense direction is functionally linked to the promoter, which already causes sterile pollen and thus sterile plants, with regard to its effect in terms of conferring male sterility, to be further enhanced.
  • an apoplastic inhibitor as also described by Rausch et al. is described (loc. cit.).
  • an intracellular invertase inhibitor linked to an apoplast (extracellular space) targeting signal can also be used.
  • large amounts can be due to the strong expression of the nucleic acids under their control or control, which is both tissue-specific (pollen and tapetum and thus anther-specific) and also temporally specific (only during pollen formation), due to the strong expression caused by the promoters according to the invention of a certain protein at a certain time and in a certain place (anthers) of transgenic plants by means of these.
  • the special protein can then be obtained in large quantities by harvesting the anthers, digestion and purification processes specific to the specific protein.
  • the promoters according to the invention and their use in transgenic plants allow intervention in the development of the anthers of plants.
  • One example is the antisense expression of invertase sequences already explained above, as a result of which the amount of extracellular invertase in the tapetum and pollen is reduced and leads to male sterile plants which are important in agriculture and in the production of hybrid seeds .
  • Hybrid seeds are of central importance for modern agriculture because they provide particularly powerful or high-yielding plants. Hybrid seeds arise from two genetically different parents, whereby the diversity of the genetic background of the parent plants is responsible for the special properties or the strengthening of the positive properties in the branch generation compared to the parent plants. This is called the heterosis effect.
  • male-sterile plants When growing plants for the purpose of producing such hybrid plants, it must therefore be avoided that there is an increase in plants with the same genetic background. In the case of separated sex plants, this can be avoided by spatial separation, although this does not guarantee the required reliability in every case.
  • the anthers of the male plants are removed manually, which is very time-consuming and particularly difficult for small-flowered plants and hermaphrodites.
  • the production of male-sterile plants is the method of choice and thus the use of the promoters according to the invention and constructs containing them.
  • the use of male sterile plants has other advantages. Because of the limited nature of the intervention, which leads to male sterility due to the use of the promoters according to the invention and the constructs containing them, the vegetative growth of the plant is not disturbed.
  • the advantages of the promoters according to the invention, the constructs and plants containing them described above for the production of hybrid seeds also apply analogously to transgenic plants which have been given certain properties using genetic engineering methods (in addition to the introduction of one of the promoters according to the invention).
  • genetic engineering methods in addition to the introduction of one of the promoters according to the invention.
  • male-sterile plants when male-sterile plants are used, there is no risk of crossing out the genetic modification (s) on plants (as a result of avoiding pollen count) that grow in neighboring fields or in the wild.
  • the special nature of the intervention i.e. the inventive development of male sterility, no interactions with the additional genetic changes to be expected.
  • the promoters according to the invention and the constructs containing them are particularly advantageous biological security systems or transgenic plants which carry this mechanism of male sterility are to be regarded as particularly safe in the sense of excluding an undesired spread of genetically modified plants.
  • the promoters according to the invention and the constructs containing them can also be used to produce transgenic plants which produce large amounts of plant-specific substances which can have a positive effect on the development of the plants, in particular the yield of fruit-bearing plants.
  • plant-specific substances would be growth hormones or protein, which are necessary for the energy supply of the growing tissues (eg invertases, sugar transporters).
  • growth hormones or protein which are necessary for the energy supply of the growing tissues (eg invertases, sugar transporters).
  • Such an increase can be caused directly by an introduced gene or directly as the result of an intervention in a control loop.
  • Auxins, cytokinins, Giberelline brassinosteroids and jasmonate can be mentioned as growth hormones, which have a predominantly stimulating effect. Abscisic acid and ethylene can be mentioned as predominantly inhibiting growth hormones.
  • the promoters according to the invention can be used not only for up-regulation of the production of compounds produced by the plant, but also for the reduction of substances produced by the plant, ie plant-specific substances (for example plant hormones) based on his knowledge, taking into account the system to be changed.
  • This reduction can be achieved by introducing degrading enzymes, inhibitors or by so-called “single-chain” antibodies.
  • Male sterility can also be reduced by means of such systems, so that a combination of this system with the systems of male production disclosed herein Sterility is possible and thus the degree of male sterility can be increased.
  • the nucleic acid construct disclosed herein according to SEQ ID. No. 8 comprising one of the promoters according to the invention and, functionally connected thereto, a nucleic acid sequence coding for an invertase introduced into a plant cell.
  • the plant cell can be any cell of a plant, in particular a leaf cell due to its totipotent character, i.e. their ability to differentiate into each type of plant cell.
  • the (plant) cell provided with the construct is then developed or regenerated into a complete plant. This plant can in turn be propagated vegetatively, for example by propagating cuttings.
  • a very similar procedure is used when male-sterile plants are generated using the system or nucleic acid construct used for the co-suppression.
  • a special form of the plants according to the invention are the so-called restorer plants. These restorer plants are required in order to maintain the yields of sterile, fruit-bearing plants, such as, for example, maize and oilseed rape, and for the multiplication of the male sterile plants according to the invention.
  • the restorer plants are distinguished by the fact that they contain a construct which leads to the production of an invertase.
  • This invertase ensures the carbohydrate supply to the anthers and thus to the tapetum and pollen.
  • the invertase is preferably a heterologous invertase of the restorer plant, ie it is different from the invertase (s) contained in the anthers, more precisely the tapetum and / or the pollen.
  • This Invertase can also be different from that / those invertases of the plant with which the restorer plant is to be crossed.
  • Invertases which are suitable in this regard are, for example, invertases of different species (for example invertases from Saccaromyces cerevisiae or from bacteria such as Zymomonas mobilis).
  • sucrose transport systems for the transport of sucrose across the cell membrane
  • intracellular enzymes that break down sucrose eg sucrose synthase or neutral or vacuolar invertases
  • sucrose synthase or neutral or vacuolar invertases can also be used for this purpose. It is crucial here that in the restorer plants according to the invention the above enzymatic activity restoring the sugar supply to the anthers is under the control of a promoter according to the invention.
  • both constructs comprising one of the promoters according to the invention are contained in a cell or a plant.
  • an antisense nucleic acid is formed which prevents the expression of the cell's own invertase and thus interrupts the sugar supply to the anthers, with the result that the pollen is sterile and the number of pollen formed is also drastically reduced .
  • the construct introduced by the restorer plant into the plant (branch generation 1), which comprises one of the promoters according to the invention and a nucleic acid for a preferably heterologous invertase or a substitute system described above, is used to maintain a sugar supply. Because the same promoter, or at least a similar promoter, is used in both constructs, the interruption of the sugar supply (due to the mechanism causing male sterility) is compensated for by the system of the restorer plant which ensures the sugar supply. This is possible because, due to the diversity of the invertorer system of the restorer plant, it is not influenced by the antisense mechanism of the male sterile plant and is therefore impaired.
  • the restorer plants of the present invention can be divided into the following three groups.
  • the first group comprises plants which, in their cells, preferably in the majority of their cells, contain one of the promoters according to the invention and a nucleic acid coding for a further invertase which is controlled by this promoter, the further invertase is different from the cell's own or anther's own or plant's own invertase.
  • the second group comprises plants which, in their cells, preferably in the majority of their cells, contain one of the promoters according to the invention and a nucleic acid coding for a sucrose transport system, this nucleic acid being controlled or controlled by this promoter.
  • a subgroup of this second group of plants comprises yet another of the promoters according to the invention which controls an additionally present nucleic acid which codes for a sucrose synthase and / or cytoplasmic expressed invertase.
  • the third group of plants are restorer plants, which combine the constructs of the first two groups.
  • the different genotypes of the parent cells combine and thus coexist the different nucleic acids coding for invertase activity or corresponding substitutes, which are preferably under the control of one of the promoters according to the invention.
  • the plants according to the invention (branch generation 1) obtained in this way are male fertile and provide the hybrid seeds which are necessary for the further occurrence of the heterosis effect and the formation of plants in which the actual crop product is the seeds, such as, for example, maize or rapeseed.
  • the sterile pollen of male-sterile plants according to the invention are locked in the mononuclear microspore stage. These pollen can now be used to cultivate haploid plants as part of an in vitro embryogenesis, which in turn can then be grown to give homozygous diploid plants. So there is a system and method are available which provide the homozygous diploid plants which are advantageous for plant breeding purposes.
  • This system is essentially made possible by the fact that in the process according to the invention for the production of male-sterile plants or from the male-sterile plants, pollen is still produced in contrast to other processes for the production of male-sterile plants in which no pollen is formed, as is the case, for example, with in the cytotoxic process for the production of male sterile plants (described in Mariani, C. et al. (1990) Induction of male sterility in plants by a chimeric ribonuclease gene. Nature 347, 737-741]).
  • Another use of the male sterile plants according to the invention is the production of seedless fruits.
  • a process for producing haploid or dihaploid, homozygous plants as an important starting material for plant breeding thus comprises the steps of obtaining the sterile pollen of a plant according to the invention and then subsequently regenerating it to haploid or dihaploid plants by in vitro embryogenesis.
  • in vitro embryogenesis is known to those skilled in the art and is described, for example, in Reynolds, T.L. (1997) Pollen embryogenesis. Plant Mol. Biology 33, 1-10.
  • a hunger and stress step is required to induce in vitro embryogenesis.
  • the sugar supply is disrupted.
  • Fig. 1 in part (A) photographically the various stages of flowering of tobacco, in part (B) the weight and length of the anthers of tobacco
  • Fig. 6 in part (A) is a photographic representation of an anther of a wt plant
  • Promoter is in part (B) pollen from anthers of transgenic tobacco plants in which a beta-glucuronidase activity is under the control of one of the promoters according to the invention, in part C, compared to B, pollen from
  • Photographs of tobacco pollen from wild-type plants and from a male sterile plant according to the invention show a representation of the pollination of different forms of the male sterile plants according to the invention; 9 shows a representation of the starch accumulation of various forms of the male sterile plants according to the invention; Fig. 10 photographs of pollen from wild-type tobacco plants and male sterile plants according to the present invention; 11 photographs of the germination behavior of pollen from
  • FIG. 12 photographs of anthers of transgenic tomatoes which contain a tobacco-derived promoter according to the invention which controls the expression of beta-glucuronidase;
  • Figure 15 shows the genomic sequence of NIN 88
  • FIG. 16 shows the construct according to the invention from the promoter according to SEQ ID No. 1 and the part of the invertase NTN 88 which is under its control and in the antisense orientation, as also shown in SEQ ID. No. 8th;
  • 17 shows the sequence of LIN 7 with annotations.
  • a 750 bp cDNA fragment of the extracellular invertase NIN 77 was cloned using reverse transcriptase of mRNA from tobacco anthers and PCR using the oligonucleotides OLN 3 and OIN 4.
  • OLN 3 and OIN 4 are primers developed for the cloning of plant invertases (Roitsch et al. (1995) Induction of apoplastic invertase of Chenopodium rubrum by D-glucose and a glucose analogue and tissue specific expression suggest a role in sink source regulation. Plant Physiol. 108, 285-294).
  • SEQ ID No. 4 The sequence of OIN 3 is referred to herein as SEQ ID No. 4 designated.
  • OIN 4 is herein SEQ ID No. 5 designated.
  • SEQ ID No. 6 The sequence of the extracellular invertase NIN 77 found in this way, or the cDNA fragment, is referred to herein as SEQ ID No. 6 designated.
  • the NIN 77 cDNA fragment was then used to screen a tobacco genomic bank in phage lambda gt 10.
  • the positive clones obtained were screened again using the ONT 4 oligonucleotide.
  • the sequence of ONT 4 is referred to herein as SEQ ID No. 7 designated
  • NIN 88 The genomic sequence of NIN 88 is found herein as Fig. 15 and as SEQ ID. No. 15.
  • Example 3 Expression analysis of the extracellular invertase NIN 88
  • the extracellular invertase NLN 88 is initially localized in cells of the endothecium and the tapetum and in a later development stage in the developing pollen, which show a continuous increase in the activity of the extracellular invertase.
  • the time sequence of the expression of NLN 88 results is illustrated in parts A to C of FIG. 3.
  • Part A of Fig. 3 shows the specific localization of the NIN 88 protein in the tapetum in an anther cross section. The detection is carried out by immunolocalization, which results in a dark border on the light bean-shaped structure inside.
  • NIN 88 can then be shown in tetrads (FIG. 3, part C) and then in the pollen (FIG. 3, part B) in an anther cross section using the antiserum described above, the detection for the tetrads in Dark-colored oval shape and the detection of the pollen in the form of round structures inside the cut light-colored bean-shaped structure.
  • Example 4 Preparation of nucleic acid constructs comprising the NIN 88 promoter.
  • the promoter was also cloned as part of the gene (composed of promoter (and other control elements) and structural gene).
  • the name of the plamide (pNDG8.3) which contains the promoter and the part of the NIN88 gene used for the NIN88 antisense construct, was also included.
  • the in SEQ ID No. 1 represents a 3 kb fragment of the NIN 88 promoter.
  • the sequence according to SEQ ID No. 1 is active as a promoter and comprises several pollen expression-specific cis-active elements according to Madison et al (1999); Plans. Mol. Biol. 41, pp. 741-751:
  • SEQ. ID No. 1 The sequence according to SEQ. ID No. 1, supplemented by approx. 1 kb at the 5 'end and provided with the above annotations, is shown in FIG. 5.
  • the compared to SEQ ID No. 1 additional 1 kb of SEQ ID No. 2 are in front of the 5 'end of SEQ ID No. 1.
  • sequence according to SEQ ID No. 1 was fused as a promoter (also referred to herein as a NIN 88 promoter) to various other coding sequences, as shown in FIG. 6.
  • the sequences were fused either in the sense orientation or in the anti-sense orientation.
  • the sense orientation leads to the formation of mRNA and thus a translation product
  • the anti-sense orientation leads to the formation of antisense nucleic acid, which interacts with sense nucleic acid and thereby prevents the formation of a translation product.
  • Invertase inhibitor is the apoplastic invertase inhibitor Nt-Inhl from tobacco described by Rausch (Greiner, S. et al. (1998) Cloning of a tobacco apoplasmic invertase inhibitor. Plant Physiol. 116, pp. 733-742]
  • the approximately 3 kb fragment of SEQ ID No. 1 as a NIN 88 promoter with the beta-glucuronidase gene as reporter gene in a derivative of the plant transformation vector pBILOl fused in sense orientation and used to transform tobacco (Nicotiana tabacum cv ( cultivar, variety). Xanthi and Samsun NN).
  • the cloning strategy is described in Example 4.3.
  • Evidence of the expression of the promoter in anthers was provided by histochemical detection of beta-glucuronidase enzyme activity on the tissues of intact anthers using the substrate X-GLUC.
  • the expression of the promoter in pollen was demonstrated both by histochemical detection of beta-glucuronidase enzyme activity and by fluorometric detection in crude extracts using the substrate MUG.
  • Part A of FIG. 6 shows the anthers which are readily histochemically stainable as a result of the expression of beta-glucuronidase.
  • Part B shows that, under the influence of the NIN 88 promoter, beta-glucuronidase can also be detected in the pollen, whereas pollen from wt plants, i.e. Wild-type plants, not stained.
  • Example 4.2 Use of the construct promoter -GUS (sense) for the transformation of tomato and Arabidopsis thaliana
  • the construct promoter - GUS (sense) described in example 4.1 was also used for the transformation of tomatoes (Lycopersicon peruvianum).
  • the specific expression of the promoter in anthers and pollen could be demonstrated by histochemical detection of the beta-glucuronidase enzyme activity. 11 shows that GUS activity can be detected specifically in the anthers of the transformed line LP 1-8, but not in the anthers of a wt plant.
  • the pollen can only be stained histochemically in the case of the transformed tomato (LP 1-8), but not in the case of the wild type, due to the presence of beta-glucuronidase activity.
  • Said construct was also used to transform Arabidopsis thaliana. As in the case of Lyopersicon, a tissue and development stage-specific expression pattern of the reporter gene was observed under the influence of the NIN 88 promoter.
  • the NIN 88 promoter was used and the sequence for the fragment of the NIN 88 (corresponds to SEQ ID No. 6) fused to it in an antisense orientation.
  • This construct is referred to herein as SEQ ID No. 8 and designated and is shown in Fig. 16.
  • Example 4.3.1 The construction of the construct according to SEQ ID. No 8 proceeds with the interposition of the promoter-beta-glucuronidase construct described in Example 4.1 as follows:
  • This construct can be used to generate the male sterile plants of the present invention by transforming them into a plant cell using known techniques and growing or regenerating the resulting transformants into whole plants. The mechanism of action involved has already been described above.
  • the construct according to SEQ ID No.8 was used to transform tobacco (Nicotiana tabacum cv. Xanthi and Samsun NN).
  • Example 4.3.2 Using the construct described in Example 4.3.1
  • the detection of the transformation was carried out by PCR using the primer NPK15 (specific for the NIN88 promoter), herein as SEQ ID No. 16, and NPK19 (specific for the NIN88 antisense construct), herein as SEQ ID No. 17 designated.
  • NT 23-81 denotes a male sterile plant which was produced using the construct according to SEQ ID. No 8, as also shown in FIG. 16.
  • the ability of the pollen to germinate is less than 1%, as can be seen from FIG. 8
  • the starch accumulation is lower, as from the finding of a negative one
  • FIG. 10 shows light microscopic images of pollen of the tobacco wild type and of a male sterile plant (NT 23-6) (produced using the construct according to SEQ ID No. 8 and FIG. 16). which support this finding and also show that the sterile pollen are less developed.
  • the pollen of the male sterile plants according to the invention does not germinate (Fig.
  • Example 4.4 Further constructs and transformations using the NLN 88 promoter
  • NPK15 corresponding to SEQ ID No. 16 (specific for the NIN88 promoter) and NPK17 (corresponding to SEQ ID No. 18) (CIN1 -antisense), NPK 18 (corresponding to SEQ ID No. 19) (invertase-inhibitor-sense) or NPK20 (corresponding to SEQ ID No. 20) (NTßfrucl -antisense).
  • CINl-antisense reduction of the germination capacity of the pollen by up to 98%
  • NTßfrucl-antisense reduction of the germination capacity of the pollen by up to 91%
  • Invertase-inhibitor-sense reduction of the germination capacity of the pollen by up to 81%
  • FIG. 13 that which is under the control of the promoter according to SEQ ID No. 3 standing extracellular invertase LIN 7 from the tomato also expressed in the anthers of the tomato.
  • RNA probe 14 here denote a negative and positive control, since a complementary, hybridizing, single-stranded antisense RNA probe is used for the specific detection of an mRNA in tissue sections by in-situ hybridization.
  • a sense probe that cannot hybridize because it has an identical base composition is used as a negative control.
  • FIG. 17 shows the sequence of LIN 7 with annotations that SEQ ID No. 3 corresponds. Sequence analysis of LIN 7 showed that this promoter carries a number of cis-acting elements that are pollen-specific:
  • CAAT-box, TATA-box and the start codon ATG are shown in bold.

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Abstract

L'invention concerne des acides nucléiques codants pour des promoteurs qui sont spécifiques du tapetum comme du pollen. L'invention concerne leur utilisation pour produire des plantes mâles stériles.
EP00949099A 1999-06-12 2000-06-13 Systeme promoteur, sa production et son utilisation Withdrawn EP1183379A2 (fr)

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DE29909998U 1999-06-12
DE29909998 1999-06-12
DE20005992 2000-04-04
DE20005992U 2000-04-04
DE20007494U 2000-04-26
DE20007494 2000-04-26
PCT/DE2000/001944 WO2000077187A2 (fr) 1999-06-12 2000-06-13 Systeme promoteur, sa production et son utilisation

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US20050120418A1 (en) * 2003-11-06 2005-06-02 Anawah Inc. Tomatoes having altered acid invertase activity due to non-transgenic alterations in acid invertase genes
CN101037695B (zh) * 2006-03-16 2011-08-17 华中农业大学 一种控制水稻花粉育性基因及应用
CN106480026B (zh) * 2016-09-29 2019-01-29 北京大学 一种花药早期发育特异性表达启动子及其应用
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