WO2002027001A2 - Plantes a fertilite masquee - Google Patents

Plantes a fertilite masquee Download PDF

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Publication number
WO2002027001A2
WO2002027001A2 PCT/DE2001/003746 DE0103746W WO0227001A2 WO 2002027001 A2 WO2002027001 A2 WO 2002027001A2 DE 0103746 W DE0103746 W DE 0103746W WO 0227001 A2 WO0227001 A2 WO 0227001A2
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nucleic acid
acid sequence
promoter
seq
sequence according
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PCT/DE2001/003746
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German (de)
English (en)
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WO2002027001A3 (fr
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Sabine Steiner-Lange
Anna Sorensen
Teresa Mozo
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to AU2002210374A priority Critical patent/AU2002210374A1/en
Priority to DE10194113T priority patent/DE10194113D2/de
Publication of WO2002027001A2 publication Critical patent/WO2002027001A2/fr
Publication of WO2002027001A3 publication Critical patent/WO2002027001A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—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
    • 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)
    • C12N15/8216—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
    • C12N15/8242—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
    • C12N15/8255—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 lignin biosynthesis

Definitions

  • the present invention relates to transgenic plants with masked fertility, the masked fertility by an altered expression of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog, or by a change in the biological activity of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog encoded gene product.
  • the present invention further relates to the regulatory nucleic acid sequence which naturally controls the expression of the MYB26 gene in Arabidopsis thaliana.
  • the present invention further relates to ner processes for producing the transgenic plants according to the invention.
  • MYB-type proteins form a superfamily of structurally similar transcription factors. MYB proteins are found in both plant and animal organisms and influence a variety of different metabolic pathways. The common structural motif of this protein family is the MYB domain, which can bind to a specific D ⁇ A sequence. The classic MYB domain consists of two or three very similar sequence repeats (repeats: Rl, R2, R3) of 50-53 AS length. Most plant-based MYB proteins have only two sequence i? Epe ⁇ t.s R2, R3). Arabidopsis has already detected 97 different R2R3-type proteins. This group forms the largest regulatory gene family known to date in plants.
  • MYB genes that have so far been characterized functionally are known to regulate, inter alia, the phenylpropanoid metabolism, to influence cell shape and differentiation, to be involved in the hormone response or to be activated in the course of plant defense reactions.
  • Summary information on the MYB family can be found in, among others, Romero et al .: More than 80 R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana. Plant J. 14, 273-284 (1998), Kranz et al: Towards functional characterization of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J. 16, 263-276 (1998) and Martin and Paz-Ares: MYB transcription factors in plants.
  • the MYB26 gene belongs to the MYB family described above.
  • a cDNA clone from Arabidopsis thaliana (Genbank Accession No. 175997) is available from this MYB26 gene, but it has not hitherto been known in which specific metabolic pathways this gene intervenes or in which tissues it is expressed.
  • a genomic clone (MDC16) is under Genbank Accession number AB019229 registered.
  • male sterile plants can be produced by different methods. In the simplest case, the male flower organs are isolated from the rest of the plant by bags or simply cut off. However, these procedures are very time, personnel and cost intensive. In addition, a large number of useful plants contain male and female organs within the same flower, which makes these processes even more difficult.
  • Male sterile plants can also be created with the help of pollen-killing or pollen-blocking chemicals (gametoids).
  • gametoids pollen-killing or pollen-blocking chemicals
  • the advantage of these chemical processes is that only temporary sterility occurs, which only persists for the duration of the treatment. However, most of these gametocides have additional phytotoxic properties. Furthermore, the treatment for plants with a long flowering phase must be repeated several times.
  • US Pat. No. 5,955,653 uses a beta-1,3-glucanase which is specifically expressed in wallpaper unicells to induce sterility.
  • US 5,962,769 achieves the same goal by increasing the endogenous avidin concentrations.
  • WO 9008830 describes the expression of protein inhibitor genes or of so-called killer genes in the male inflorescences which are intended to cause the anthers and associated tissues to die off.
  • WO 90/08831 seeks a similar effect through the expression of disruptor genes which prevent mitochondrial cell respiration.
  • WO 89/10396 very generally proposes to transform the plant genome with exogenous so-called “male sterility DNAs” (for example coding for noses, doses, proteases, lipases etc.), which, when expressed in cells of the stamens, metabolism, function or interfere with the development of this cell.
  • EP 0329308 describes antisense DNAs for influencing the Pollen production used.
  • the hybrids are often characterized by uniformity, increased growth, higher yield or increased resistance to pathogens. This effect, known as heterosis, is of great commercial interest for agricultural businesses or plant breeders and requires targeted crossings.
  • Male sterile plants facilitate these crossbreeding techniques because they are easier to obtain hybrid offspring.
  • male sterile and male fertile plants are planted side by side in one field.
  • the male sterile plants can only be fertilized by pollen from the male fertile plants and accordingly only produce hybrid seeds. It is particularly advantageous if the sterility is not permanent, as in most known sterile mutants, but is reversible, so that e.g. controlled self-fertilization attempts will be possible as part of later selection and characterization processes.
  • the object of the present invention is therefore to provide novel male sterile plants and processes for their production.
  • Figure 1 shows the promoter region of the Arabidopsis thaliana MYB26 gene. The start ATG is highlighted in bold.
  • Figure 2 shows the genomic nucleic acid sequence of the Arabidopsis thaliana MYB26 gene. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns. The numbering continues the numbering from FIG. 1.
  • Figure 3 shows the amino acid sequence of MYB26 derived from the open reading frame of Figure 1.
  • Figure 4 shows a Southern analysis of individuals on line 7AAB12.
  • DNA from 14 F2 progeny from 7AAB12 [five plants that do not carry a mutant allele for andl (1-5), four heterozygous plants for andl (6-9) and five sterile plants (10-14)] were digested with EcoRI separated on a 0.8% agarose gel and transferred to a nylon membrane.
  • the left photo shows the hybridization with the left end of the transposon.
  • Each individual shows 6-10 bands, of which only one with a size of 3.8 kb occurs exclusively in plants with at least one mutated allele from andl, whereas they are absent in the plants which carry the wild type allele of this locus homozygously.
  • the corresponding gang is marked with an arrow.
  • the right photo shows the hybridization of the same membrane with a MYB26-specific probe (without MYB domain).
  • the 3.8 kb band which occurs in conjunction with the mutation in andl, also hybridizes with AtMYB26.
  • Plants that carry the andl wild type allele homozygously (1-5) show a band of 6.3 kb, which corresponds to the wild type focus of AtMYB26. Both bands occur in andl-heterozygous plants (6-9) with approximately the same intensity.
  • a weak signal at the level of the wild-type band in sterile plants can be explained by the transposon jumping out in some cells of the plant (somatic sectors).
  • FIG. 5 shows the original sequence of the N-terminus of the MYB26 protein compared to the corresponding sequence of a stable mutant.
  • the first line shows the N-terminus of the MYB26 protein.
  • the original en insertion site is marked.
  • the N-terminus of the MYB26 protein is shown after the transposon did not jump out.
  • Three amino acids were deleted.
  • FIG. 6 shows a microscopic view of a cross section through anther tissue in MYB26 mutants (left) and wild type (right). While radial cell wall thickenings (arrow) can be seen in the wild type, these are absent in the MYB26 mutant.
  • FIG. 7 shows a male sterile plant of the Arabidopsis thaliana species with a gene defect in the MYB26 gene in comparison with the wild type plant.
  • masked fertility used here denotes a non-release of the pollen from the pollen sacs as a result of a morphological and cellular change in the pollen sac tissue, as a result of which the plants concerned are male-sterile.
  • homologs or “homologous sequences” used here denote nucleic acid or amino acid sequences with significant similarity to the comparison sequence or parts thereof. Homologous sequences are thus nucleic acid sequences which hybridize with the comparison sequences or parts of these sequences under stringent or less stringent conditions (for stringent and less stringent conditions see Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory (1989), ISBN 0-87969- 309-6).
  • stringent hybridization conditions is: Hybridization in 4 x SSC at 65 ° C (alternatively in 50% formamide and 4 X SSC at 42 ° C), followed by several washing steps in 0.1 x SSC at 65 ° C for a total of about one Hour.
  • the homologous sequences are further to be considered nucleic acid or amino acid sequences or parts thereof which, with the aid of the BLAST similarity algorithm (Basic Local Alignment Search Tool, Altschul et al, Journal of Molecular Biology 215, 403-410 (1990)) have a significant similarity to comparison sequences Sequences are described as significantly similar, as used here, which, for example using standard parameters in the BLAST service of the NCBI, have a significance level (E-Value or Probability) of P ⁇ 10 "5 if they are compared with the comparison sequences.
  • BLAST similarity algorithm Basic Local Alignment Search Tool, Altschul et al, Journal of Molecular Biology 215, 403-410 (1990)
  • derivatives used here denotes nucleic acid sequences which, compared to the comparison sequence, have modifications in the form of one or more deletions, substitutions, additions, insertions and / or inversions. Introns are also derivatives containing genomic equivalents of EST or cDNA sequences. Derivative also means that a nucleic acid sequence is composed of one or more nucleic acid fragments of a gene and one or more nucleic acid fragments of one or more other genes. The individual domains can be both unmodified and modified.
  • fragments denotes parts of amino acid sequences or their genetic information in the form of nucleic acids, provided that they have at least one functionally important region (domain, sequence or structure motif) of the comparison sequence, such as, for example, catalytic centers or motifs interacting with proteins, furthermore Elements of DNA binding such as a MYB DNA binding domain repeat signature 1 (Prosite Accession number PS00037) MYB_1 with the consensus sequence W- [ST] -x (2) -E- [DE] -x (2) - [LIV ] or a MYB DNA-binding domain repeat signature 2 (Prosite Accession number PS00334) MYB_2 with the consensus sequence Wx (2) - [LI] - [SAG] -x (4,5) -Rx (8) - [YW] - x (3) - [LIVM]
  • the corresponding database entries come from the PROSITE database (Hofmann et al .: The PROSITE database, its Status in 1999. Nucleic Acids Res.
  • a regulatory sequence such as a promoter controls the expression of a gene or another functional nucleic acid or that a nucleic acid sequence is expressed starting from the promoter.
  • nucleic acid or “functional nucleic acid sequence” as used herein means a nucleic acid sequence that does not encode a naturally occurring gene product, e.g. a ribozyme, an antisense DNA or RNA or a sequence composed of different exons.
  • vector used here denotes naturally occurring or artificially created constructs for the uptake, multiplication, expression or transfer of nucleic acids, for example plasmids, phagemids, cosmids, artificial chromosomes, bacteriophages, viruses, retroviruses.
  • transgenic plant used here relates to plants which were produced by means of recombinant genetic engineering and / or microbiological processes and not by means of conventional breeding processes.
  • expression system means any combination of vectors, restriction enzymes, transformation methods, cell extracts, living cells e.g. prokaryotic or eukaryotic cells or organisms for the purpose of endogenous or exogenous expression of genes.
  • One aspect of the present invention relates to transgenic plants with masked fertility, the masked fertility by an altered expression of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog compared to wild type plants, or by changing the biological activity of the by the nucleic acid sequence according to SEQ ID NO: 2 or whose homologously encoded gene product is produced.
  • the present invention further relates to methods for producing the transgenic plants according to the invention.
  • Another aspect of the present invention relates to the use of the Nucleic acid sequence according to SEQ ID NO: 2 or its homolog, derivative or fragment for the production of plants with an altered lignin content, the altered lignin content being caused by an expression changed compared to wild type plants, or by changing the biological activity of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog, derivative or fragment encoded gene product.
  • changed expression here means that the expression can be increased, reduced or completely switched off. Accordingly, in the sense of the invention, a changed lignin content means that the lignin content is increased or decreased.
  • Enhanced expression can e.g. by combining the nucleic acid sequence according to SEQ ID NO: 2 or its homolog with a strong promoter.
  • suitable promoters which can lead to an increased constitutive expression of the MYB26 gene or its homolog are the CaMV 35S promoter from the cauliflower mosaic virus and the ubiquitin promoter from maize.
  • either the endogenous promoter which controls the expression of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog can be replaced by the strong promoter, or an additional copy of the nucleic acid sequence according to SEQ ID NO: 2 or its homologue can be inserted into the genome can be integrated, the additional copy being functionally linked to the strong promoter.
  • Increased expression can also be achieved by integrating more than one additional copy of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog, in particular two, three, four, five or more copies, into the genome. These copies can be integrated individually or contiguously. Furthermore, the copies are functionally linked to a promoter, which can be a strong promoter, an endogenous or an exogenous promoter that controls the expression of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog, or a tissue-specific or inducible promoter.
  • a promoter which can be a strong promoter, an endogenous or an exogenous promoter that controls the expression of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog, or a tissue-specific or inducible promoter.
  • Constructs comprising a corresponding promoter and a nucleic acid sequence according to SEQ ID NO: 2, or its homolog, which is functionally linked thereto, can be used by conventional methods for the transformation of plant cells and subsequently for the regeneration of complete plants.
  • the introduction of nucleic acid sequences into plant organisms and cells is state of the art and used, for example of T-DNA vectors, easy to do.
  • a multiple of the proteins encoded by these nucleic acid sequences is correspondingly formed.
  • Enhanced gene expression of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog can also be achieved by changing or adding functional components of the promoter which naturally regulates the expression of the MYB26 gene.
  • corresponding functional components are TATA box, CAAT box, GC box, enhancer or binding sites for other transcription factors.
  • Suitable methods for changing the nucleic acid sequence of the promoter which naturally regulates the expression of the MYB26 gene are known to the person skilled in the art.
  • Known mutagenesis techniques are, for example, the generation of deletion mutants or the introduction of point mutations by "site directed mutagenesis".
  • Preferred methods for mutagenesis or for changing the expression are furthermore chimeric RNA / DNA oligonucleotides, homologous recombination or RNA interference (RNAi).
  • Transgenic plants that produce an increased amount of MYB26 transcripts or their homologues compared to the wild type due to increased gene expression or additional MYB26 gene copies have a severely disturbed MYB26 gene balance. Due to the change in tissue metabolism in the pollen sac, the function of the anthers is no longer ensured, so that masked fertility occurs.
  • a reduced expression of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog can be achieved by changing or removing essential functional components of the promoter which naturally regulates the expression of the MYB26 gene.
  • corresponding components are TATA-Box, CAAT-Box, GC-Box, enhancers or binding sites for other transcription factors. Suitable processes for this are known to the person skilled in the art.
  • Known mutagenesis techniques are, for example, the generation of deletion mutants or the introduction of point mutations by "site directed mutagenesis”.
  • Preferred methods are also chimeric RNA / DNA oligonucleotides, homologous recombination or RNA interference (RNAi).
  • Transgenic plants with a reduced MYB26 gene expression only produce small amounts of the transcription factor necessary for the formation of secondary cell wall thickenings in the anthers. By reducing the corresponding lignifications, the anthers can no longer open, resulting in masked fertility.
  • a complete deactivation of the gene expression of the nucleic acid sequence according to SEQ ID NO: 2 or its homolog can be achieved by introducing new start or stop codons or by shifting the reading frame. As a result of the changed nucleic acid sequence, functionally inactive proteins are synthesized.
  • Preferred methods for a targeted gene knockout of the MYB26 gene are the introduction of point mutations by "site directed mutagenesis”.
  • Preferred methods for mutagenesis or for changing the expression are furthermore chimeric RNA / DNA oligonucleotides, homologous recombination or RNA interference (RNAi).
  • Transgenic plants with MYB26 gene knockout have anthers without lignifications of the endothelial cell wall, as are characteristic of the wild type. After the pollen ripens, the anther walls do not recede, which results in masked fertility.
  • antisense copies of the MYB26 nucleic acid sequence and their fragments, homologs or derivatives can also be stably integrated into the genome. Due to the reverse orientation of the antisense nucleic acid sequence relative to the promoter, antisense RNAs are formed during transcription, which form duplex molecules with the naturally formed MYB26 mRNAs and thus prevent translation.
  • the corresponding technology is established and e.g. in Izant and Weintraub: Inhibition of thymidine kinase gene expression -by antisense RNA: a molecular approach to genetic analysis. Cell 36, 1007-1015 (1984).
  • the antisense gene or its fragment, homolog or derivative can e.g. are placed under the control of an inducible promoter, whereby the production of antisense RNA can be regulated.
  • a change in the biological activity of the gene product encoded by the nucleic acid sequence according to SEQ ID NO: 2 or its homolog can be achieved by introducing modifications in gene segments which code for functional protein domains such as, for example, receptor binding sites, phosphorylation domains or DNA binding sites. Modifications in the MYB-DNA binding domain in particular have a considerable influence on the properties of the protein. In this way, proteins can be formed that are, for example, inactive are, have a changed substrate specificity or a changed Km value or are no longer subject to the regulatory mechanisms normally present in the cell via allosteric regulation or covalent modification. Correspondingly modified MYB26 proteins are no longer able to perform the physiological functions of the MYB26 wild type protein.
  • Suitable methods for changing the sequence of the MYB26 gene are known to the person skilled in the art.
  • Known mutagenesis techniques are, for example, the generation of deletion mutants or the introduction of point mutations by "site directed mutagenesis”.
  • Preferred methods for mutagenesis or for changing the expression are furthermore chimeric RNA / DNA oligonucleotides, homologous recombination or RNA interference (RNAi).
  • RNAi RNA interference
  • the MYB26 genes introduced into the genome can be the nucleic acid sequence according to SEQ ID NO: 2 or its fragment or homolog or derivative, for example any MYB26 sequence from other plants.
  • the nucleic acids used can be of natural origin or have been produced artificially.
  • the nucleic acid sequences can be in either sense or antisense orientation.
  • the regulatory DNA sequence functionally linked to the nucleic acid sequence according to SEQ ID NO: 2 or its fragment or homolog or derivative, for example a promoter can be the regulatory DNA sequence according to SEQ ID NO: 1 endogenously present with the nucleic acid sequence as well as any other regulatory DNA sequence.
  • the regulatory DNA sequence can be either an endogenous promoter of the organism to be transformed or an exogenous promoter which is located, for example, on the vector used.
  • any regulatory sequence that can control the expression of foreign genes in organisms, for example plants is suitable as a promoter, for example the CaMV 35S promoter from the cauliflower mosaic virus; see. Franck et al., Cell 21, 285-294 (1980) or the ubiquitin promoter from corn.
  • the expression of the nucleic acid sequences can also be achieved by a chemically inducible promoter.
  • chemically inducible promoters are the PRPI promoter (Ward et al., Plant Molecular Biology 22, 361-366 (1993)), a promoter induced by salicylic acid (WO 95/19443), a promoter inducible by benzenesulfonamide (EP-A 388186), a promoter inducible by tetracycline (Gatz et al., Plant Journal 2, 397-404 (1992)), a promoter inducible by abscisic acid (EP-A 335528) or by Ethanol or cyclohexanone inducible promoter (WO 93/21334).
  • Promoters can also be used which are active, for example, in certain plant tissues or parts of plants.
  • Examples of corresponding promoters are the phaseolin promoter (US 5504200), the isoflavone reductase promoter (US 5750399), a seed-specific promoter, for example from tobacco (US 5824863) or the ST-LSI promoter from potato (Stockhaus et al., (1989) EMBO J ' 8, 2445-2452).
  • the regulatory DNA sequence functionally linked to the nucleic acid sequence according to SEQ ID NO: 2 or its fragment or homolog or derivative for producing a transgenic plant with an altered lignin content can, for. B. be a promoter, the tissue-specific z. B. in the phloem or vascular tissue of plants leads to a strong expression of the nucleic acid sequences functionally linked to it.
  • promoters are, for example, the glutamine synthase promoter (Edwards et al. (1990) Proc. Natl. Acad. Sci. USA 87: 3459-3463), the maize sucrose synthetase 1 promoter (Yang et al. ( Ward 93) Proc. Natl. Acad.
  • the nucleic acid sequences used can be endogenous or exogenous nucleic acid sequences or their fragments or derivatives or homologues.
  • Endogenous means that the nucleic acid sequence comes from the same organism into which it is integrated with the method according to the invention, e.g. a MYB26 gene from Arabidopsis thaliana is integrated into Arabidopsis thaliana using the method according to the invention.
  • Exogenous means that the nucleic acid sequence comes from another organism, e.g. a MYB26 gene from Arabidopsis thaliana is described in e.g. Wheat integrated.
  • nucleic acid sequences compared to the naturally occurring nucleic acid sequences deletions, substitutions, additions, Lisertions and / or inversions.
  • the present invention further relates to a transformed cell, in particular a transformed plant cell or a transformed plant tissue, in which a regulatory Nucleic acid sequence, for example the nucleic acid sequence according to the invention according to SEQ ID NO: 1, and the nucleic acid sequence according to SEQ ID NO: 2, or their fragments or homologs or derivatives, are stably integrated.
  • the present invention also relates to a plant cell or a transformed plant cell or a transformed plant tissue transformed with the regulatory nucleic acid sequence, for example the nucleic acid sequence according to the invention according to SEQ ID NO: 1, and the nucleic acid sequence according to SEQ ID NO: 2, or its fragments or homologues or derivatives can be regenerated into a seed-producing plant.
  • the present invention relates to a plant which can be obtained by the process according to the invention.
  • the present invention further relates to seed obtained from plants obtained by the process according to the invention.
  • the invention further relates to the fruits, seeds, leaves, shoots and storage organs produced by the plants, for example fruits, berries, grapes, cereals and potatoes.
  • a nucleic acid sequence is used which is composed of nucleic acid fragments from different MYB26 genes.
  • This composite nucleic acid sequence can also contain modifications such as deletions, additions or substitutions.
  • the methods for producing such nucleic acid sequences are state of the art and can be easily carried out by a person skilled in the art.
  • the present invention can be applied to any plant organism.
  • the present invention provides male sterile mono- and dicotyledonous plants.
  • Particularly preferred plants are cultivated plants such as soybean, cotton, flax, hemp, sunflower, potato, tobacco, tomato, zucchini, eggplant, cucumber, summer rape, winter rape, alfalfa, lettuce, chicory, asparagus, salsify, pea, bean, lentils, carrot , Onion, leek, olive, radish, radish, beetroot, turnip, kohlrabi, sugar beet, beet, sugar cane, spinach, chard, the different tree, nut and wine species, the different types of cabbage such as Brussels sprouts, cauliflower, broccoli, white cabbage, red cabbage , Savoy cabbage, Chinese cabbage, also cereals such as barley, wheat, rye, oats, corn, rice, forage grasses, furthermore types of fruit such as mango, apple, pear, peach, plum, raspberry, blackberry, gooseberry, strawberry
  • the plants transformed with the nucleic acid sequence can be unmodified wild-type plants or plants obtained by breeding or modified plants, for example transgenic plants.
  • the invention can also be used in plant tissues and in plant cells, for example in a cell culture or in expression systems, to change the expression pattern of MYB26 genes or fragments or derivatives or homologues of these genes.
  • the method according to the invention is suitable for temporary sterilization by exchanging the MYB26-specific promoter for an inducible promoter. Through targeted induction of fertility e.g. be reacted to special environmental conditions.
  • the present invention further relates to vectors comprising a nucleic acid sequence according to SEQ ID NO: 2, or its fragment or derivative or homolog and a regulatory DNA sequence.
  • Suitable vectors for the uptake and transfer of the nucleic acid sequences can be the multiplication and / or the expression of the uptake nucleic acids in single cells such as e.g. Ensure Escherichia coli or Agrobacterium tumefaciens or in plant cells, plant tissues or plants. Corresponding vectors can of course occur or can be produced artificially.
  • the vectors can include selection markers, terminator sequences, polylinkers, promoter elements, enhancers, polyadenylation sites and other genetic elements.
  • Vectors suitable for cloning are e.g.
  • Plasmids of the pBluescript series, plasmids of the pUC series, plasmids of the pGEM series or vectors based on the bacteriophage ⁇ are e.g. pBinl9; see. Bevan et al., (1984), Nucleic Acids Research 12, 8711-8721.
  • vectors which can be used on the Ti plasmid of Agrobacterium species or constructs based on plant viruses are usable and are known to the person skilled in the art.
  • Transformation methods for the transfer of foreign genes (transformation) into the genome of plants are presented below.
  • the choice of the method for introducing the nucleic acid sequence according to the invention and the nucleic acid sequences which are functionally linked thereto and which code for a gene product into plant cells is not restricted to this list. Transformation methods used to date in plants are, for example, gene transfer using Agrobacterium tumefaciens (e.g.
  • Another aspect of the present invention relates to the regulatory nucleic acid sequence (hereinafter also referred to as promoter), which naturally controls the expression of the MYB26 gene in Arabidopsis thaliana.
  • This nucleic acid sequence according to the invention is listed in SEQ ID NO: 1.
  • the invention further relates to fragments or homologs or derivatives of the nucleic acid sequence according to SEQ ID NO: 1, which have the biological function of a promoter, preferably a promoter active in the male flower organs.
  • the invention further relates to nucleic acid sequences which hybridize with the nucleic acid sequence according to SEQ ID NO: 1 and which have the biological form of a promoter, preferably a promoter active in the male flower organs.
  • Preferred are nucleic acid sequences which hybridize under stringent conditions with the nucleic acid sequence according to SEQ ID NO: 1 and which have the biological form of a promoter, preferably a promoter active in the male flower organs.
  • the regulatory nucleic acid sequence according to SEQ ID NO: 1 or its fragment or homolog or derivative is suitable, for example, for identifying and isolating genes homologous to the MYB26 gene in other organisms and / or regulatory sequences related to SEQ ID NO: 1 in, for example, Arabidopsis thaliana or other organisms using special hybridization or screening methods, for example as a probe for screening in DNA libraries with the help of hybridization to single-stranded nucleic acids Base sequence.
  • the regulatory nucleic acid sequence according to SEQ ID NO: 1 or its fragment or homolog or derivative is also suitable for the specific control of the expression of genes or other functional nucleic acids in organisms or cells, due to their tissue specificity preferably for the specific control of genes in the male flower organs.
  • tissue specificity preferably for the specific control of genes in the male flower organs.
  • the genes or other functional nucleic acids functionally linked to the regulatory nucleic acid sequence according to SEQ ID NO: 1 or its fragment or homolog or derivative can be endogenous, exogenous genomic DNA segments or cDNAs or their fragments or derivatives or synthetic or semisynthetic nucleic acids.
  • Endogenous means that the nucleic acid sequence comes from the same organism into which it is integrated with the method according to the invention, e.g. a nucleic acid sequence from Arabidopsis thaliana is integrated into Arabidopsis thaliana using the method according to the invention.
  • Exogenous means that the nucleic acid sequence comes from another organism, e.g.
  • nucleic acid sequence from Arabidopsis thaliana is obtained with the method according to the invention in e.g. Wheat integrated.
  • the functionally linked genes or other functional nucleic acid sequences can have deletions, substitutions, additions, iterations and / or inversions compared to the naturally occurring nucleic acid sequences.
  • nucleic acid sequence according to SEQ ID NO: 1 or its fragment or homolog or derivative can be used for regulating the expression of the MYB26 gene which is naturally associated with it, so that plants with increased, reduced or completely absent MYB26 expression are present, which affects the MYB26 metabolic pathway and consequently leads to a structural change in the pollen sac tissue and the associated masked fertility.
  • the regulatory nucleic acid sequence according to the invention is also suitable for the Regulation of expression of other gene sequences.
  • the promoter can be present in combination with any genes, both in a vector and in transgenic organisms.
  • the promoter can be used for the regulation of the expression of genes in the flower organs, particularly for the regulation of the expression of genes in the anthers, especially in the pollen sac tissue.
  • genes from all plant metabolic pathways can be regulated by the promoter, for example genes coding for components of fat metabolism (e.g. acyl-CoA synthase, acetyl-CoA carboxylase etc.), carbohydrate metabolism (e.g.
  • phosphofructokinase fructose-1, 6-bisphosphatase
  • Glucose-6-phosphate dehydrogenase etc. amino acid metabolism (e.g. aminotransferases, deaminases etc.), hormone metabolism (e.g. genes for the synthesis of auxins, gibberellins, abscisic acid etc.), photosynthesis (e.g. Rieske protein, psaE etc.) , Cell architecture (e.g. tubulin, actin etc.), respiration (e.g. cytochrome C, cytochrome C oxidase etc.), defense against pathogens (flavonoids, terpenes etc.) etc. etc.
  • amino acid metabolism e.g. aminotransferases, deaminases etc.
  • hormone metabolism e.g. genes for the synthesis of auxins, gibberellins, abscisic acid etc.
  • photosynthesis e.g. Rieske protein, ps
  • Particularly suitable genes to be regulated influence e.g. structure, size or metabolism of the pollen sac, so that transgenic plants with modified male flower organs, eg larger, more stable or shape-changing pollen sacs, can arise.
  • Corresponding genes can be involved in the construction of pollen sac structures, for example, in the synthesis or incorporation of lignin or cellulose for secondary cell wall thickening.
  • An example of a corresponding anther-specific gene is the CHSLK gene (Genbank Accession Number Y14506), presumably involved in lignin synthesis, coding for a putative chalcone synthase.
  • Genes for the enzymes that modify or degrade the cell wall structures can also be mentioned here, for example A6, a gene for an anther-specific ⁇ -1,3-glucanase (Genbank Accession number X62716). Further examples of anther-specific genes with partially still unknown functions are sf2 (Genbank accession number X62716), RA8 (Genbank accession number AF042275) ATA20 (Genbank accession number AF037362) and MZm3-3 (Genbank accession number AJ224355). Also particularly suitable for regulation with the aid of the promoter are genes which, when specifically expressed in the male flower organs, also produce male sterility in plants, for example as a result of the death of the affected cells. A wide variety of proteins can be expressed for this purpose, which significantly disrupt the normal metabolism of the cell. Kataboh enzymes such as nucleases, proteases, lipases or glucanases are particularly suitable for this.
  • the regulatory nucleic acid sequences according to the invention according to SEQ ID NO: 1 or their fragments or homologs or derivatives can be of natural origin or have been produced artificially.
  • the functionally linked genes or the other functional nucleic acid sequences can be in either sense or antisense orientation.
  • the regulatory nucleic acid sequence according to SEQ ID NO: 1 or its fragments or homologs or derivatives can be used in vectors, expression systems or plants, plant tissues or plant cells or animal cells or microorganisms to change the expression patterns of a wide variety of gene products.
  • the expression of the gene products can be both increased and reduced compared to their natural expression.
  • the present invention accordingly also relates to vectors comprising a regulatory nucleic acid sequence according to SEQ ID NO: 1 or its fragment or derivative or homolog.
  • Suitable vectors for the uptake and transfer of the nucleic acid sequences can ensure the multiplication and / or the expression of the uptake nucleic acids in single cells such as, for example, Escherichia coli or Agrobacterium tumefaciens or in plant cells, plant tissues or plants.
  • Corresponding vectors can of course occur or can be produced artificially.
  • the vectors can include selection markers, terminator sequences, polylinkers, promoter elements, enhancers, polyadenylation sites and other genetic elements.
  • Vectors suitable for cloning are, for example, plasmids of the pBluescript series, plasmids of the pUC series, plasmids of the pGEM series or vectors based on the bacteriophage ⁇ .
  • a plasmid vector used for use in Agrobacterium is, for example, pBin19; see. Bevan et al., (1984), Nucleic Acids Research 12, 8711-8721.
  • vectors which can be used on the Ti plasmid of Agrobacterium species or constructs based on plant viruses are usable and are known to the person skilled in the art.
  • transformation methods such as microinjection, protoplast transformation and microprojectile bombardment also use the abovementioned cloning vectors or linearized DNA instead of Ti plasmids.
  • a summary description of vectors used to date can be found in Guerineau and MuUineaux, Plant Transformation and Expression Vectors, in: Plant Molecular Biology Labfax, published by Croy, Oxford, BIOS Scientific Publishers, 121-148 (1993).
  • Some of the transformation methods used in commercial transformation and expression systems for the transfer of foreign genes (transformation) into the genome of plants are presented below.
  • the choice of the method for introducing the nucleic acid sequence according to the invention and the nucleic acid sequences which are functionally linked thereto and which code for a gene product into plant cells is not restricted to this list.
  • Transformation methods used to date in plants are, for example, gene transfer using Agrobacterium tumefaciens (e.g. by bathing seeds, inflorescences or leaf fragments in an agrobacterial solution), using plant viruses, by electroporation, by injecting (microprojectile bombardment) or by injecting (microinjection) and incubating dry ones Embryos in DNA-containing liquids and the transformation of protoplasts with the help of polyethylene glycol. More detailed descriptions of the methods mentioned can be found, for example, in Jens et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by Kung and Wu, Academic Press 128-143 (1993).
  • the invention also relates to transgenic plants with a regulatory nucleic acid sequence according to SEQ ED NO: 1 which is stably integrated into the genome, or its fragment or derivative or homolog with the biological function of a promoter, preferably a promoter active in the male flower organs, and one with this nucleic acid sequence functionally linked for a gene product or another functional nucleic acid sequence corresponding to the examples of such coding or other functional nucleic acid sequences listed and described above.
  • a promoter preferably a promoter active in the male flower organs
  • the present invention further relates to a method for producing a plant with modified gene expression, comprising the stable integration of a regulatory sequence according to SEQ ID NO: 1 or its fragment or homolog or derivative with the biological function of a promoter, preferably a promoter active in the male flower organs , and a nucleic acid sequence coding for a gene product or another functional nucleic acid sequence that is functionally linked to this sequence into the genome of plant cells or plant tissues and regeneration of the plant cells or plant tissues obtained to plants.
  • the method according to the invention can be applied to any plant organisms.
  • the method according to the invention can be used to change the gene expression of mono- and dicotyledonous plants.
  • Particularly preferred plants are cultivated plants such as soybean, cotton, flax, hemp, sunflower, potato, tobacco, tomato, zucchini, eggplant, cucumber, summer rape, winter rape, alfalfa, lettuce, chicory, asparagus, salsify, pea, bean, lentils, carrot , Onion, leek, olive, radish, radish, beetroot, turnip, kohlrabi, sugar beet, beet, sugar cane, spinach, chard, the different tree, nut and wine species, the different types of cabbage such as Brussels sprouts, cauliflower, broccoli, white cabbage, red cabbage , Savoy cabbage, Chinese cabbage, also cereals such as barley, wheat, rye, oats, corn, rice, forage grasses, furthermore types of fruit such as mango, apple, pear, peach, plum, raspberry, blackberry, gooseberry, strawberry, cherry, currant, guava, banana , Melon, pumpkin and citrus fruits eg lemon, orange, grapefruit or tangerine
  • the plants transformed with the nucleic acid sequence can be unmodified wild-type plants or plants obtained by breeding or modified plants, for example transgenic plants.
  • the method according to the invention can also be used in plant tissues and in plant cells, for example in a cell culture or in expression systems, to change the expression pattern of MYB26 genes or fragments or derivatives or homologues of these genes.
  • cloning steps carried out in the context of the present invention such as e.g. Restriction cleavage, agarose gel electrophoresis, purification of nucleic acid fragments, transfer of nucleic acids to filter materials, transformation and cultivation of bacterial cells, etc. were performed as in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989), ISBN 0-87969-309-6.
  • Example 2 Preparation of a knockout population of Arabidopsis thaliana The present invention was obtained by screening a knockout population mutagenized with the transposon En-1 / Spm (Pereira et al, EMBO Journal 5, 835-841 (1986)) from plants of the species Arabidopsis thaliana Ecotyp Columbia. The integration of the transposable elements in genes of the mother plant often leads to the failure of the corresponding gene function and in many cases to a wild-type appearance of the affected plant. To build up the knockout population, the autonomous En-1 element from Zea mays was transferred to Arabidopsis using Agrobacterium tumefaciens.
  • the corresponding transposon tagging system is described in Cardon et al., Plant Molecular Biology 23, 157-178 (1993).
  • the Ti plasmid used, pGV3850HPT :: pkEn2 contained the complete En-1 element as an integrate.
  • this vector carries the HPT gene under the control of the viral CaMV 35S promoter. Seeds of a transformant with a single T-DNA insert were sown on hygromycin-containing medium. Seeds of the resulting hygromycin-resistant plants (T 2 generation) were then sown on a medium containing kanamycin. In the plants selected in this way (T 3 generation), the Enl element was transposed out of the T-DNA.
  • T 4 generation Those plants of the T 4 generation which carried one or more transposed En-1 elements but no longer any En-1 elements in the T-DNA were identified by means of PCR. However, these plants still contained the T-DNAs without integrated En-1 elements.
  • T-DNA negative plants To generate En-1 positive, T-DNA negative plants, the T 4 generation was crossed with the wild-type Arabidopis thaliana ecotype Columbia and En-1 positive, T-DNA negative plants (S 0 generation) were identified by PCR.
  • the mutant phenotype is linked to the homozygous state of a particular integration of the transposons.
  • 18 sister plants of the en line 7AAB12 were analyzed. Six of these plants were sterile, indicating a recessive mutation. Phenotypic analysis of 18 offspring each of the fertile individuals showed that seven of the 18 parent plants also produced sterile offspring and were therefore heterozygous for andl, whereas five plants showed no sterile offspring and were therefore homozygous for the wild type allele.
  • flanking DNA from eight different en insertions was obtained by “transposon insertion display” (TID), cf. Yephremov and Saedler, The Plant Journal 21, 495-505, 2000, and cloned into the pGemTeasy vector (company Promega)
  • TID transposon insertion display
  • cf. Yephremov and Saedler The Plant Journal 21, 495-505, 2000
  • cloned into the pGemTeasy vector company Promega
  • the inserts of the resulting plasmids were sequenced. Sequence comparisons with sequence databases yielded 100% agreement with genomic Arabidopsis sequences in five cases.
  • the lengths of restriction fragments calculated from the sequence were compared with the fragments obtained in Southern blot analyzes.
  • a sequence shows agreement between calculated fragment lengths with the experimentally determined lengths for the band which occurs coupled with the andl mutation.
  • This sequence of the PI clone MDC16 bears in the region of the transposon integration site an open reading frame which has 100% identity to the cDNA of the putative transcription factor AfMYB26 the AtMYB26 cDNA (without MYB dom ane) showed that all sterile plants homozygously carry an En insertion in AtMYB26, whereas all plants which do not carry the mutation, and also have no insertion in AtMYB26.
  • the fifth oldest still closed bud of wild type and MYB26 mutant was fixed in 2% paraformaldehyde / 0.25% glutaraldehyde for 2 h and then embedded in paraffin. Semi-thin sections were stained with Calcofluor White as described in Dawson et al., 1999 (New Phytologist 144: 213-222).

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Abstract

L'invention concerne des plantes transgéniques à fertilité masquée provoquée par l'expression modifiée de la séquence d'acide nucléique conformément à SEQ ID NO:2 ou son homologue, ou par la modification de l'activité biologique du produit génétique codé par la séquence d'acide nucléique conformément à SEQ ID NO: 2 ou son homologue. La présente invention porte également sur la séquence d'acide nucléique régulatrice qui, dans Arabidopsis thaliana régule de manière naturelle l'expression du gène MYB26. Ladite invention concerne également des procédés pour produire lesdites plantes transgéniques.
PCT/DE2001/003746 2000-09-28 2001-09-28 Plantes a fertilite masquee WO2002027001A2 (fr)

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AUPO947997A0 (en) * 1997-09-26 1997-10-23 Commonwealth Scientific And Industrial Research Organisation Method of regulating gene expression
US6559357B1 (en) * 1999-01-08 2003-05-06 The Regents Of The University Of California Methods for altering mass and fertility in plants
WO2000056907A1 (fr) * 1999-03-22 2000-09-28 Institute Of Molecular Agrobiology Regulation de la formation des sporocytes ou meiocytes chez les plantes
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KRANZ HARALD D ET AL: "Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana." PLANT JOURNAL, Bd. 16, Nr. 2, Oktober 1998 (1998-10), Seiten 263-276, XP002193001 ISSN: 0960-7412 *
MEISSNER RUTH C ET AL: "Function search in a large transcription factor gene family in Arabidopsis: Assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes." PLANT CELL, Bd. 11, Nr. 10, Oktober 1999 (1999-10), Seiten 1827-1840, XP002192999 ISSN: 1040-4651 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1765058A1 (fr) * 2004-06-15 2007-03-28 Latrobe University Molecules d'acides nucleiques et utilisation de celles-ci dans la sterilite des vegetaux
EP1765058A4 (fr) * 2004-06-15 2008-05-28 Univ Latrobe Molecules d'acides nucleiques et utilisation de celles-ci dans la sterilite des vegetaux
EP2241624A3 (fr) * 2004-06-15 2011-02-09 Latrobe University Molécules d'acides nucléiques et utilisation de celles-ci dans la stérilite des végétaux
US8163975B2 (en) 2004-06-15 2012-04-24 La Trobe University Nucleic acid molecules and their use in plant sterility
US8624086B2 (en) 2004-06-15 2014-01-07 La Trobe University Nucleic acid molecules and their use in plant sterility

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