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WO2000029566A1 - Promoteurs d'expression genique dans les racines de plantes - Google Patents

Promoteurs d'expression genique dans les racines de plantes Download PDF

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Publication number
WO2000029566A1
WO2000029566A1 PCT/EP1999/008786 EP9908786W WO0029566A1 WO 2000029566 A1 WO2000029566 A1 WO 2000029566A1 EP 9908786 W EP9908786 W EP 9908786W WO 0029566 A1 WO0029566 A1 WO 0029566A1
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Prior art keywords
seq
promoter
plants
plant
root
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PCT/EP1999/008786
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German (de)
English (en)
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Jörg Riesmeier
Lothar Willmitzer
Marcel Bucher
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ETH Zürich
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Priority to JP2000582550A priority Critical patent/JP2002530075A/ja
Priority to AU16518/00A priority patent/AU1651800A/en
Priority to EP99959279A priority patent/EP1135480A1/fr
Priority to CA002350186A priority patent/CA2350186A1/fr
Publication of WO2000029566A1 publication Critical patent/WO2000029566A1/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)
    • C12N15/8216—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
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific
    • 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

Definitions

  • the present invention relates to promoters which bring about a root-specific expression of coding nucleotide sequences controlled by them for tissue-specific gene expression in plants, expression cassettes, recombinant vectors and microorganisms which comprise such promoters, thus transformed transgenic plants, a process for producing transgenic plants and a process for Isolation of root-specific promoters.
  • promoters are of great importance for the production of transgenic plants, since the specificity of a promoter is decisive for the point in time, in which tissue types and in what intensity a genetically transmitted structural gene is expressed.
  • a large number of promoters which control the expression of foreign genes in plants are known.
  • the most frequently used promoter is the 35S CaMV promoter (Franck et al, Cell 1 (1980), 285-294), which leads to a constitutive expression of the introduced gene.
  • inducible promoters are also frequently used, for example for wound induction (DE-A-3843628), chemical induction (Ward et al., Plant Molec. Biol.
  • promoters which mediate gene expression in root are the class H patatin promoter (Koester-Toepfer et al., Mol. Gen. Genet. 219 (1989), 390-396), which after fusion with the reporter gene of the ⁇ -glucuronidase (GUS) gene shows a high expression in potato tubers and in certain cell layers of root tips.
  • GUS fusion experiments with the agropine synthase promoter (ags) (Inoguchi et al., Plant Phys. 149 (1996), 73-78) also showed high GUS activity, especially in roots.
  • promoters described are often problematic. Promoters which bring about a consumer expression of the genes controlled by them can be used, for example, to produce herbicide-tolerant and pathogen-resistant plants but the disadvantage that the products of the genes they control in all parts of the
  • Plant present even in the harvested parts of the plant, which can be undesirable in some cases.
  • Inducible promoters are also not without problems, since the induction conditions in agricultural plants in the field are typically difficult to control.
  • genes that are to be regulated differently are required under the
  • the present invention is therefore based on the object of providing agents which enable an organ-specific, preferably root-specific, gene expression of plants. These agents should be suitable, for example, for the expression of genes which influence the uptake of nutrients from the soil and of genes which modify the root growth.
  • the present invention thus relates to a promoter selected from the group consisting of a) promoters which are listed under Seq ID No. 1, SEQ ED No. 2, SEQ ID No. 3, SEQ ED No. 4, SEQ ID No. 5 or SEQ ID No. Include 6 nucleic acid sequence given; b) promoters that contain a functional part of the Seq ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 comprise the nucleic acid sequence indicated and which bring about a root-specific expression in plants of a coding nucleotide sequence controlled by them; c) promoters which have a sequence which corresponds to one of those listed under Seq ID No.
  • the promoters according to the invention are derived from or from plant genes.
  • a “promoter” is understood to mean a DNA sequence which comprises the regulatory portion of a gene, preferably a structural gene.
  • the "regulatory part” of a gene is understood to mean that part which determines the expression of the gene.
  • a regulatory part has a sequence motif on which transcription factors and RNA polymerase assemble and initiate the transcription of the coding part of the gene.
  • the regulatory component can include one or more positive regulatory elements, so-called enhancers. In addition or instead of this, it can also contain negative regulatory elements, so-called silencers.
  • a “structural gene” is understood to mean a genetic unit composed of a regulatory and coding part, the gene product of which is a protein.
  • the information for the primary amino acid sequence of the protein is contained in the coding portion of the structural gene, while the regulatory portion determines when and in what tissues the amounts of the transcript of the coding portion are formed, after the presentation of which the gene product is synthesized.
  • the promoters according to the invention can originate, for example, from plant genes, be modified by recombinant DNA techniques and / or be produced synthetically.
  • the term “root-specific” is understood to mean that a foreign gene under the control of a promoter according to the invention in the
  • Roots is expressed. In particular, root specificity is within the scope of the present
  • Foreign gene in the roots favored in comparison to mature leaves and in roots caused a significantly increased expression, for example at least 2 to 5 times, preferably 5 to 10 times, particularly preferably 10 to 100 times.
  • root specificity can be achieved by
  • the promoter can be used, for example, in a
  • Reporter genes such as the ß-glucuronidase gene from E. coli. This
  • the various promoter fragments are preferably constructed in such a way that they do not encompass this 5 'upstream area (items 1-255, SEQ ED No. 7).
  • the promoter according to the invention now allows root-specific gene expression of a coding nucleotide sequence controlled by the promoter. It is an interesting alternative to other root-specific promoters because it can also mediate gene expression in root hairs. Because of the larger surface area of the root hair compared to the root, there is the possibility that the expression of those genes whose gene products mediate, for example, the transport of nutrients and metabolites through the root hair cells can be manipulated more effectively with the aid of the promoter according to the invention.
  • the promoter according to the invention can be used for the root-specific expression of those genes which mediate the absorption of, for example, heavy metal ions from contaminated soils. With the help of the promoter according to the invention it would thus be possible to produce transgenic plants which can be used in phytoremediation.
  • the promoters according to the invention allow the expression of a coding nucleotide sequence controlled by them in specific root cells, such as for example in root cells of the primary root, in root hairs of the Primary root, in root cells of the root tip or in root hairs of the primary root below the hypocotyl.
  • the present invention also relates to promoters which have a functional part of this sequence and which bring about a root-specific expression of a coding nucleotide sequence controlled by them in plants.
  • a "functional part” is understood to mean those sequences which, despite a different nucleotide sequence, still have the desired functions, such as e.g. Have promoter activity and tissue or organ specificity.
  • a measure of the promoter activity is, for example, the expression rate determined for a specific marker gene which is under the regulatory control of the promoter according to the invention.
  • suitable marker genes are the ⁇ -glucuronidase (GUS) gene from E. coli or the green fluorescence protein (GFP) gene (Baulcombe et al., Plant J. 7 (16) (1993), 1045 - 1053).
  • GUS ⁇ -glucuronidase
  • GFP green fluorescence protein
  • Functional parts of the promoter sequences within the scope of the present invention include naturally occurring variants of the sequences described here as well as artificial, e.g. nucleotide sequences obtained by chemical synthesis.
  • a functional part is also understood to mean, in particular, natural or artificial mutations in an originally isolated promoter sequence which continue to show the desired functions. Mutations include substitutions, additions, deletions, exchanges and / or insertions of one or more nucleotide residues.
  • the present invention also encompasses those nucleotide sequences which can be obtained by modifying the sequence shown under Seq ED No. 1, SEQ ID No. 2, SEQ ED No. 3, SEQ ED No. 4, SEQ ID No. 5 or SEQ ED No. 6 nucleotide sequence specified can be obtained. The aim of such a modification can e.g. further narrowing down the promoter sequence contained therein or e.g. also be the insertion of further restriction interfaces.
  • Functional parts of a promoter sequence also include those promoter variants whose promoter activity is weakened or enhanced compared to the wild type.
  • the activity of a eukaryotic RNA polymerase II promoter is caused by the synergistic interaction of various tr ⁇ «5-active factors (DNA binding proteins) which bind to the various cis-regulatory DNA elements present in the promoter. These factors interact directly or indirectly with single or multiple factors of the basal transcription machinery, which ultimately leads to the formation of a pre-initiation complex in the vicinity of the transcription start site (Drapkin et al., Current Opinion in Cell Biology 5 (1993), 469-476).
  • RNA polymerase II promoters eukaryotic RNA polymerase II promoters
  • different cw elements modules
  • This modular structure was illustrated, for example, by promoter studies with the cauliflower mosaic virus (CaMV) 35S promoter (Benfey and Chua, Science 250 (1990), 959-966; Benfey et al., EMBO J. 9 (1990) , 1677-1684; Benfey et al, EMBO J. 9 (1990), 1685-1696).
  • CaMV cauliflower mosaic virus
  • the promoter was divided into 6 subdomains.
  • the strong, constitutive expression mediated by the full promoter can thus be sectioned into tissue-specific sub-activities.
  • a minimal promoter is understood to mean a DNA sequence, a TATA box which is approximately 20 to 30 base pairs upstream from the transcription start site, or an initiator sequence (Smale and Baltimore, Cell 57 (1989), 103-113; Zawel and Reinberg , Proc. Natl. Acad. Sci. 44 (1993), 67-108; Conaway and Conaway, Annu. Rev. Biochem 62 (1993), 161-190).
  • minimal promoters are, for example, the -63 to +8 ⁇ 35S promoter (Frohberg, dissertation at the FU Berlin FB Biologie (1994)), the -332 to +14 minimal Patatin Classl promoter and the -176 to + 4- Minimal PetE promoter (Pwee et al, Plant J. 3 (1993), 437-449.)
  • subdomains or cts elements of the promoter according to the invention can also be identified by deletion analyzes or mutagenesis (Kawagoe et al, Plant J. 5 (6) (1994), 885-890).
  • the test for functionality of such a subdomain or -Elements can be transformed in planta by detecting the reporter gene activity
  • the functional part of the promoter sequence also means those subdomains or cw elements of the SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No.
  • ED No. 6 indicated nucleotide sequences that impart root specificity.
  • the effectiveness of a subdomain or a c ⁇ element can be significantly increased by multimerization.
  • the CaMV-35S promoter for example, dimerization of a 250 bp fragment in tandem arrangement resulted in a tenfold increase in promoter activity (Kay et al., Science 230 (1987), 1299-1302).
  • subdomain B5 of the CaMV-35S promoter there was a significant increase in activity of the promoter construct when this domain was present as a tetramer and not as a monomer (Benfey et al, EMBO J. 9 (1990), 1685-1696).
  • the present invention therefore also relates in particular to dimers and multimers of subdomains or cw elements of the SEQ ID No. 1, SEQ ID No. 2, SEQ ED No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 indicated nucleotide sequences.
  • the increase in promoter activity compared to the wild type is achieved by combining the promoter according to the invention with a so-called enhancer.
  • tissue specificity being generally determined by the enhancer used in each case (Benfey et al, Science 250 (1990), 959-966; Benfey et al, EMBO J. 8 (1989), 2195-2202; Chen et al, EMBO J. 7, (1988), 297-302; Simpson et al., Nature 323 (1986), 551-554).
  • enhancers such as the PetE enhancer (Sandhu et al., Plant Mol. Biol. 37 (1998), 885-896), which do not have a tissue-specific effect and can therefore be placed in front of the promoter according to the invention as purely quantitative enhancer elements, in order to increase expression in roots without changing the quality of the tissue specificity of the promoter according to the invention.
  • a root-specific enhancer based on the multimerization of a specific box box
  • synthetic enhancers can also be used which are derived, for example, from naturally occurring enhancers and / or by combining different enhancers
  • the present invention also relates to promoters which have a nucleotide sequence which is identical to that shown under SEQ ID no. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 nucleotide sequence shown hybridize and which cause a root-specific expression of a coding nucleotide sequence controlled by them in plants.
  • Such sequences preferably hybridize under stringent conditions with that under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ED No. 4, SEQ ID No. 5 or SEQ ID No. 6 sequence shown.
  • stringent conditions preferably means hybridization conditions, as described, for example, in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). In particular, hybridization takes place under the following conditions:
  • Such promoters preferably have a sequence identity of at least 30%, preferably at least 40%, preferably at least 50%, particularly preferably at least 60%, particularly preferably at least 70% and advantageously at least 80%, preferably at least 90% and particularly preferably at least 95% o to that under Seq ID No. 1 promoter sequence shown or parts thereof.
  • the sequence identity of such promoter sequences is preferably determined by comparison with the one under SEQ ID No. 1 nucleotide sequence shown determined. If two sequences to be compared have different lengths, the
  • the parameters are preferably set so that the
  • Percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of nucleotides in the
  • SEQ ED No. 4 hybridize shown nucleotide sequence or a sequence identity to SEQ ID No. 1, preferably come from vegetable
  • Organisms preferably from higher plants, particularly preferably from dicotyledonous plants, particularly preferably from plants of the Lycopersicon genus.
  • the promoter according to the invention has all of the SEQ ID no. 3 (approx. 1.4 kb fragment), SEQ ID No. 4 (approx. 1.1 kb fragment), SEQ ED No. 5 (0.9 kb fragment) or SEQ ID No. 6 (0.55 kb fragment) shown sequence.
  • the present invention also relates to promoters which have a functional part of these sequences and which bring about a root-specific expression in plants of a coding nucleotide sequence controlled by them.
  • the promoter according to the invention has all or part of the functional part of SEQ ID no. 4 (1.1 kb fragment) sequence shown.
  • SEQ ID No. 1 SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 promoter shown comes from a plant gene belonging to a group of extensin-like proteins.
  • the present invention thus also relates to promoters of genes which encode a protein, preferably from the group of the extensin-like proteins, and which have a homology, i.e. Identity, of at least 60%, preferably of at least 70%, preferably of at least 80%, particularly preferably of at least 90% and in particular of at least 95% of the total under SEQ ID No. 8 indicated amino acid sequence, these promoters in plants causing a root-specific expression of a coding nucleotide sequence controlled by them.
  • a homology i.e. Identity
  • the present invention relates to promoters of genes that contain a protein with the SEQ ID no. Coding 8 given amino acid sequence, these promoters cause a root-specific expression of a nucleotide sequence controlled by them in plants.
  • the under SEQ ID No. The nucleotide sequence shown in Figure 7 encodes a polypeptide (SEQ ID No. 8) from L. esculentum which can be assumed to belong to the group of extensin-like proteins.
  • an extensin-like protein is understood to mean a plant protein whose amino acid sequence has at least one of the following sequence motifs: SPPPPP, SPPPPYY, SPPPPY, SPPPPVY, PPPPPYY, PPPPPSY, PPPPPTY, PPPPPAY, PPPPPEY, PPPPPPPPP, PPPPPPPP, PPPPPPPP , SPPPPPPPP , SPPPPKKPYYPP, SPPPPSP, SPPPPSPKYVYK, SPPPPSPSPPPP, SPPPPYYH, SPPPPYYYK, SOOOOTOVYK, SPPPPTPVYK, SOOOOVYK, SPPPPPPYP, SPPPPVYPPVPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPP
  • DNA sequences the homology to that under SEQ ID No. 7, can be identified, for example, by computer-aided sequence comparisons with known sequences, or also by screening, for example, cDNA or genomic libraries with the SEQ ID no. 7 shown sequence or parts thereof. Such techniques are known to those skilled in the art (see e.g. Sambrook et al., Op. Cit.). Likewise, computer-aided sequence comparisons can also be carried out at the amino acid level with the sequence shown in SEQ ID No. 8 specified amino acid sequence or parts thereof.
  • the cell wall influences both the shape and the function of the cell.
  • the protein fraction of the cell wall contains both enzymes and structural proteins.
  • the best characterized structural proteins of the cell wall include the so-called extensions (Cassab and Varner, Annu Rev Plant Physiol Plant Mol Biol 39 (1988), 321-353; Showalter, Plant Cell 5 (1993), 9-23.) Extensines belong to the Family of hydroxyproline-rich glycoproteins (HRGPs). Genes and cDNAs coding for extensins have so far been characterized from carrot (Chen and Varner, EMBO J. 4 (1985), 2145-2151), Bohne (Corbin et al., Mol. Cell Biol.
  • Rapeseed (Evans et al., Mol. Gen. Genet. 223 (1990), 273-287), tomato (Showalter et al., Plant Mol. Biol. 16 (1991), 547-565) and tobacco (Memelink et al, EMBO J. 6 (1987), 3579-3583).
  • the gene expression of the extensine is development-dependent and is tissue-specific rather than constitutively regulated (Sommer-Knudsen et al., Phytochemistry 47 (1998), 483-497; Ye et al, Plant Cell 3 (1991), 23-37). Comparing the tissue specificity of different extensin genes from different plants, it is found that the expression pattern of extensin genes can vary significantly depending on the type of plant examined, the cell type and the tissue type (Showalter et al, Plant Mol. Biol. 19 (1992) , 205-215). In soybean, HRGPs are most strongly expressed in meristematic cells.
  • the HRGPnt3 gene from tobacco is expressed in the pericyclic and in the endodermis, especially in certain cells which are involved in the formation of side roots (Keller et al., Proc. Nat. Acad. Sei 86 (1989), 1529).
  • extensin gene expression can be regulated by environmental factors such as e.g. Light, pathogen infestation, wounding, heat stress (see for example Cassab and Varner, Annu Rev. Plant Physiol. Plant Mol. Biol. 39 (1988), 321-353; Cortin et al., Mol. Cell Biol. 7 (1987), 4337 -4344; Niebel et al., Plant Cell 5 (1993), 1697-1710).
  • environmental factors such as e.g. Light, pathogen infestation, wounding, heat stress (see for example Cassab and Varner, Annu Rev. Plant Physiol. Plant Mol. Biol. 39 (1988), 321-353; Cortin et al., Mol. Cell Biol. 7 (1987), 4337 -4344; Niebel et al., Plant Cell 5 (1993), 1697-1710).
  • this is not the case for all extensines (Sommer-Knudsen et al, Phytochemistry 47 (1998), 483-497).
  • the mature extensin protein is usually rich in hydroxyproline (Hyp), serine and certain combinations of the amino acids valine, tyrosine, lysine and histidine.
  • the primary structure of extensins (for an overview see, for example, Sommer-Knudsen, Phytochemistry 47 (1998), 483-497) is usually characterized by at least one Ser-Pro 3.6 peptide unit, which can also be repeated or in conjunction with similar sequences, such as can, for example, the following sequence motifs SOOOO, SPPPPKH, SPPPPPPKK, SPPPPKKPYYPP, SPPPPSP, SPPPPSPKYVYK, SPPPPSPSPPPP, SPPPPYYYH, SPPPPYYYK, SOOOOTOVYK, SPPPPTPVYK, SOOOOVYK, SPPPPVYK, SPPPPVYSPPPP, SPPPPVHSPPPPPPVA, SPPPPVK, SPPPPVKSPPPP, SOOOOVK
  • Extensines are subject to a strong post-translational modification.
  • proline residues are hydroxylated by prolyl hydroxylases.
  • the hydroxyproline residues serve as attack sites for glycosylations (Lamport et al, Biochem J. 133 (1972), 125-132; van Holst, Plant Physiol. 74 (1984), 247-251).
  • the carbohydrates primarily galactose and arabinose (Smith et al., Phytochemistry 25 (1986), 1021ff; Holst et al., Plant Physiol.
  • the prolyl hydroxylase can be selectively inhibited so that the biosynthesis of the hydroxyproline can be reduced with the help of Dhp.
  • Carrot root discs (Daucus carota) have been shown to treat structurally modified HRGPS after treatment with Dhp. The treatment of tobacco protoplasts with Dhp also led to the regeneration of cell walls with an altered structure (Cooper, Plant Physiol. 104 (1994), 747-752).
  • the present invention further relates to expression cassettes containing a promoter according to the invention.
  • expression cassette is understood to mean the combination of a promoter according to the invention with a nucleic acid sequence to be expressed.
  • This nucleic acid sequence can be, for example, a sequence encoding a polypeptide, ie a structural gene. It can be linked to the promoter in sense or antisense orientation.
  • the nucleic acid sequence can also encode a non-translatable RNA, for example an antisense RNA or a ribozyme. These nucleic acid sequences can be used in conjunction with the promoter according to the invention to produce plants with an altered, preferably improved phenotype.
  • the metabolism of the plant in the root can be influenced by means of the promoter according to the invention.
  • Some examples of heterologous (over) expression and antisense inhibition with the aim of manipulating metabolic flows in transgenic plants are summarized in Herbers and Sonnewald (TIBTECH 14 (1996), 198-205).
  • An example of ribozymes was published by Feyter (Mol. Gen. Genet. 250 (1996), 329-228).
  • a variety of possible uses of transgenic plants which can be generated with the aid of the promoters and vectors according to the invention are also described in TIPTEC Plant Product & Crop Biotechnology 13 (1995), 312-397.
  • the expression cassettes according to the invention can also be a
  • transcription termination sequence downstream of the 3 ' end of the nucleic acid sequence linked to the promoter.
  • a "transcription termination sequence” is understood to mean a DNA sequence which is at the 3 'end of a coding sequence
  • An example of such a termination sequence is that of the octopine synthase gene.
  • one or more can be used between the promoter and the nucleic acid sequence or between the nucleic acid sequence and the terminator
  • the present invention relates to vectors which contain at least one promoter according to the invention.
  • the promoter according to the invention is linked in such a vector to a polylinker which allows integration of any sequences downstream of the promoter.
  • a “polylinker” is understood to mean a DNA sequence which contains recognition sequences of at least one restriction enzyme, preferably two or more restriction enzymes.
  • a vector according to the invention also contains a sequence for the termination of the transcription, for example that of the octopine synthase gene, downstream of the promoter or the polylinker.
  • the present invention also relates to vectors which contain expression cassettes according to the invention.
  • the vectors according to the invention advantageously contain selection markers which are suitable for identifying and, if appropriate, selecting cells which contain the vectors according to the invention.
  • the vectors according to the invention are suitable for transforming plant cells and particularly preferably for integrating foreign DNA into the plant genome.
  • An example of such vectors are binary vectors, some of which are already commercially available.
  • the present invention further relates to host cells which are genetically modified with a promoter according to the invention or with an expression cassette or vector according to the invention.
  • Genetically modified means that the host cell contains a promoter or an expression cassette or vector according to the invention, preferably stably integrated into the genome, and the promoter or expression cassette was either introduced into the host cell or into a predecessor of this cell as foreign DNA . That the cells according to the invention can either themselves be the direct product of a transformation event or cells derived therefrom which contain a promoter according to the invention or an expression cassette according to the invention. Both prokaryotic, in particular bacterial, and eukaryotic cells can be considered as host cells. Eukaryotic cells can be, for example, fungal cells, in particular of the Saccharomyces genus, preferably of the Saccharomyces cerevisiae species.
  • the invention relates to the use of vectors according to the invention, expression cassettes according to the invention or host cells according to the invention for the transformation of plants, plant cells, plant tissues or parts.
  • the host cells according to the invention are plant cells, which are referred to below as transgenic plant cells.
  • the present invention also relates to plants which contain plant cells according to the invention. These can belong to any plant type, genus, family, order or class. Both monocot and dicot plants can be used.
  • the plants according to the invention are preferably useful plants, ie plants which are of agricultural, forestry and / or horticultural interest for humans. Agricultural crops such as cereals (for example wheat, oats, barley, rye), maize, rice, potatoes, beets, tobacco, sugar cane, sugar beet, sunflower, banana, rape or fodder are preferred and pasture grasses (such as alfalfa, white clover, red clover), flax, cotton. Soy, millet,
  • Beans, peas etc, vegetables such as tomato, cucumber, zucchini, eggplant,
  • Herbs and medicinal plants are also of interest, e.g. Catharanthus roseus, Datura stramonium, Taxus SSP.I, Dioscorea deltoidea, Papaver somniferum, Atropa belladonna,
  • the present invention also relates to processes for the production of transgenic plants, characterized in that plant cells, tissues or parts or protoplasts are transformed with a vector according to the invention or with an expression cassette according to the invention or with a microorganism according to the invention, the transformed cells, Tissues, parts of plants or protoplasts are cultivated in a growth medium and, if appropriate, regenerated from the plants.
  • the invention relates to the use of vectors according to the invention, expression cassettes according to the invention or host cells according to the invention for producing transgenic hairy roots by Agrobacterium rhizogenes.
  • plants of the invention can be prepared by methods known to those skilled in the art, e.g. by transforming plant cells or tissues and
  • Electroporation of DNA the introduction of DNA using the biolistic approach and other possibilities.
  • Plants preferably using the Agrobacterium-mediated transformation, have been studied intensively and are sufficient in EP 0 120 516; Hoekema (In: The Binary Plant
  • Protoplast transformation the electroporation of partially permeabilized cells or the introduction of DNA using glass fibers.
  • the transformation of maize in particular is described several times in the literature (e.g. WO 95/06128, EP 0 513 849, EP 0 465
  • the present invention also relates to propagation and cultivation material of plants according to the invention which contains plant cells according to the invention.
  • the term "propagation material" includes those components of the plant that are suitable for the production of progeny by a vegetative or generative route.
  • vegetative propagation for example, cuttings, callus cultures, rhizomes, rhizomes or tubers are suitable.
  • Other propagation material includes, for example, fruits, seeds, seedlings, protoplasts, cell cultures, etc.
  • the propagation material is preferably tubers and seeds.
  • the present invention further relates to a method for identifying and isolating promoters which bring about a root-specific expression in plants of a coding nucleotide sequence controlled by them, comprising the following steps: a) Hybridization of a plant genomic library with a cDNA which codes for an extensin-like protein ; b) isolation of positive clones; c) Testing the isolated clones for promoter activity.
  • the hybridization carried out in step a) is preferably carried out under stringent conditions.
  • Known sequences which encode an extensin-like protein or parts thereof are used for hybridization with a corresponding library, preferably under stringent conditions. Preference is given to SEQ ED No. 7 listed sequence or parts thereof used. The methods are known to the specialist and are described in detail, e.g. in Sambrook et al. (op. cit.).
  • an extensin-like protein means a protein whose amino acid sequence has at least one of the sequence motifs SPPPPP, SPPPPYY, SPPPPY, SPPPPVY, PPPPPYY, PPPPPSY, PPPPPTY, PPPPPAY, PPPPPEY, PPPPPPPPY, PPPPOP SPPPPKK, SPPPPKKPYYPP.
  • SPPPPVYSPPPP SPPPPVHSPPPPVA, SPPPPVK, SPPPPVKSPPPP, SOOOOVKP and that has a homology of at least 60%>, preferably at least 70% > , preferably at least 80% o, particularly preferably at least 90% and in particular at least 95%> to that below SEQ ID No. 7 indicated coding region.
  • the promoter sequence is carried out according to methods which are known to the person skilled in the art and which are described, for example, in Sambrook et al. (op. cit.).
  • the expression properties of the isolated promoter can be analyzed by reporter gene experiments.
  • the promoter can be operated, for example, in an expression cassette or in a vector for plant transformation with a reporter gene, such as e.g. the ß-glucuronidase gene from E. coli. This construct is used to transform plants.
  • the organ-specific expression of the ⁇ -glucuronidase is then determined, e.g. in Martin et al. (The GUS Reporter System as a Tool to Study Plant Gene Expression, In: GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, Academic Press (1992), 23-43). Those promoters that have root specificity are selected and then isolated.
  • an operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements, each of the elements mentioned being able to fulfill its function as intended for gene expression.
  • the present invention relates to the use of the promoters according to the invention or the promoters identified by means of the method according to the invention for the root-specific expression of transgenes in plants.
  • transgene means a DNA sequence artificially introduced into a plant.
  • Figure 1 Northern blot analysis of RNA from roots
  • RNA was isolated using the hot phenol extraction method of Verwoerd et al. (Nucleic Acids Research 17 (1989), 2362).
  • RNA was separated electrophoretically, transferred to a nylon membrane and fixed thereon with UV radiation. After hybridization and washing, the radioactive membrane was exposed on X-ray film. This was developed after an overnight exposure at -80 ° C. The intensity of the signals correlates with the concentration of the specific mRNA on the membrane.
  • Figure 2 Northern blot analysis for the expression of LeExtl 10 ⁇ g total RNA (5 ⁇ g in the case of lanes 3 and 4: 18 days and earth) from 7, 14 and 18
  • RNA "Northern blot" analysis Day old seedlings (7 days, 14 days, 18 days), roots grown on soil (soil), seedling roots which were incubated in the dark (light) or in light (+ light), and controls (control), as well as wounded leaves (Wound) were used for the RNA "Northern blot" analysis. Radioactive labeled LeExtl cDNA was used for hybridization. Plant RNA was isolated according to Verwoerd et al. (1989) (see above). The RNA was separated electrophoretically, transferred to a nylon membrane and fixed thereon with UV radiation. After hybridization and washing, the radioactive membrane was exposed on X-ray film. This was developed after an overnight exposure at -80 ° C. The intensity of the signals correlates with the concentration of the specific mRNA on the membrane.
  • LeExtl transcript in tomato roots Optical micrographs of young tomato roots under sterile conditions.
  • A, B Sections embedded in paraffin were made according to the sztw hybridization method as in Daram et al. (Planta 206 (1998), 225-233) described hybridized with non-radioactively labeled LeExtl "antisense" RNA.
  • Example 1 Isolation of a root-specific gene
  • RNA from the specified organs was determined by the method of Verwoerd et al. (Nucleic Acid Research 17 (1989), 2362). 7-60 ⁇ g of total RNA from root hair was used for the isolation of the poly (A) + RNA using magnetic oligodT beads (Dynabeads, Dynal, Germany). 700 ng poly (A) + RNA were used for the production of double-stranded cDNA (Pharmacia, Germany). After attaching an Ec ⁇ RI / Notl linker, the cDNA was cloned into the expression vector ⁇ ZAPII (Stratagene, Germany). Packaging in the ⁇ phages was carried out with the Gigapack II Gold Packaging ⁇ xtract Kit from Stratagene (Germany).
  • RNA from the specified organs were loaded onto a 1.2% agarose glyoxal gel (Hüll (1985), Purification, biophysical and biochemical characterization of viruses with special reference to plant viruses. In: Mahy, (ed.), Virology, A Practical Approach, IRL Press, Oxford, UK, pp. 1-24). "Northern blot" analysis and hybridization conditions were taken from standard instructions (Sambrook et al., See above). The blots were hybridized at 68 ° C. Detection of LeExtl mRNA was achieved using the LeExtl cDNA as a radioactive probe.
  • the blot was last washed with 0.1 x SSC at 68 ° C. After washing, the blots were exposed on X-ray film (Kodak, Germany) at -80 ° C. The transcript sizes were determined by comparison with glyoxylated marker DNA (BRL, Germany). The results are shown in FIGS. 1 and 2.
  • Example 2 Isolation of a Genomic Fragment Comprising the Promoter Region of the Root Specific Gene
  • a tomato genomic DNA library (Clontech Laboratories, USA) was screened with radioactively labeled LeExtl cDNA (see Example 1).
  • 500,000 plaque-forming units (PFE) from the DNA library were plated on YT agar in agar dishes, incubated for 6 to 8 hours at 37 ° C. and transferred to nylon membranes (Hybond N, Amersham, Germany) (Sambrook et al., See above). Hybridization conditions were taken from standard regulations (Sambrook et al., See above).
  • 30 plaques from the first screening were selected for a second round. The phage suspensions of two selected plaques after the second screening were replated to produce phage lysates.
  • ⁇ DNA was prepared according to Lockett (Analytical Biochemistry 185, (1990), 230-234). Each 600 ng ⁇ DNA was then digested with various restriction enzymes and the resulting fragments made visible on an agarose gel with ethidium bromide.
  • a total of three different types of GUS expression cassettes were produced.
  • the genomic fragment was excised from the pBluescript II SK " plasmid by Kpnl digestion, smoothed by means of a replenishment reaction with the T7 DNA polymerase (Ausubel et al., Current Protocols in Molecular Biology.
  • pBI101.3 is a plasmid which contains a promoterless GUS cassette in the binary vector pBIN19.
  • a PCR product of approx. 3270 bp was produced using the following primers: 42Xba (5'-GAGATCTAGACCATGGAGAAGAATTGG-3 ⁇ SEQ ID No. 11) and 42Sal (5'-GAGAGTCGACGGGCGAATTGGGTACCG-3 ', SEQ ID No. 12) .
  • the PCR conditions were: 20 sec at 94 ° C. melting of the DNA, 45 sec at 50 ° C. installation of the primers, 2 min at 72 ° C. DNA synthesis using Pfu DNA polymerase (Stratagene, Germany).
  • the PCR product was cloned directly into the plasmid pCR-Script according to the manufacturer's instructions (Stratagene, Germany).
  • the PCR product was smoothed with the Klenow enzyme [Sambrook, 1989 # 68] and cloned into Sm ⁇ l and dephosphorylated pBluescript II SK " plasmid.
  • the plasmid was digested with BstXI and Linear shortening of the PCR product using exonuclease III and S1 nuclease from the 5 'end in accordance with the manufacturer's protocol (Fermentas, Lithuania) produced shortened genomic fragments of the following approximate length: 2.2 kb, 1.7 kb, 1.4 kb, 1.1 kb , 0.9 kb and 0.55 kb.
  • the AKI ⁇ -GUS-3'NOS cassette from the plasmid 5 ⁇ KT1-320.X (Lagarde et al, Plant J. 9 (1996), 195-203) was isolated with Sacl, by means of mung bean nuclease Treatment smoothed (New England BioLabs Inc., Bioconcept, Switzerland) and digested again with Ncol
  • the resulting GUS-3' ⁇ OS cassette was digested in NcoI / EcoRV and dephosphorylated pBluescript II SK " plasmids containing the truncated genomic fragments kloni ert.
  • the promoter-GUS-3'NOS cassettes thus produced were isolated from the respective plasmid by digestion with Sacl and S ⁇ I and digested and dephosphorylated binary vector Binl9 in Sac ⁇ / Sa (Bevan et al, Nucleic Acids Research 12 (1984), 8711) cloned.
  • the contracts obtained were called BIN- ⁇ genx-GUS in the following, where x is the respective fragment length 2.2 kb (SEQ ED No. 1), 1.7 kb (SEQ ID No. 2), 1.4 kb (SEQ ID No. 3), 1.1 kb (SEQ ED No. 4), 0.9 kb (SEQ ED No. 5) or 0.55 kb (SEQ ED No. 6).
  • Example 4 Plant transformation
  • Agrobacteria were grown on YEB medium (Vervliet et al., Journal of General Virology 26
  • the tobacco and tomato plants were transformed by the method of gene transfer mediated by Agrobacterium tumefaciens, as by
  • Rosahl et al. (EMBO J. 6 (1987), 1155-1159) for tobacco and by Lillatti et al.
  • constructs described above were furthermore introduced by electroporation into the A. rhizogenes strain 15834 (Jung et al., Biotechnol Lett 14 (1992), 695-700) and then for the transformation of Catharanthus roseus by means of Agrobacterium rhizogenes based on Toivonen et al. (Plant Cell Tissue Org. Cult. 18 (1988), 79 ff.).
  • the plant material was vacuum-filtered with a 0.1% X-Gluc solution (pre-dissolve 0.1 g of X-Gluc in 1 ml of dimethylformamide, 1 ml of 10% Triton and 5 ml of 1 M sodium phosphate buffer, pH 7.2 and make up to 100 ml with still water) and incubated overnight at 37 ° C. After staining, the plants were fixed in 3: 1 ethanoic acetic acid and decolored in 100% ethanol. Green parts of the plant became colorless, while the blue dye remains stable; see Figure 3.
  • X-Gluc solution pre-dissolve 0.1 g of X-Gluc in 1 ml of dimethylformamide, 1 ml of 10% Triton and 5 ml of 1 M sodium phosphate buffer, pH 7.2 and make up to 100 ml with still water
  • the GUS activity in leaves was compared to that in roots according to the method described by Jefferson et al. (EMBO J. 6 (1987), 3901-3907).
  • GUS activity in roots is between 20 and 300 times higher than in mature leaves.
  • Most of the GUS-positive potato plants examined showed strong GUS activity in the roots, but no GUS activity in mature leaves.
  • the GUS activities were compared to those of the 35S promoter. The lines with the strongest activity of the promoter had about half as much activity compared to the CaMV 35S promoter.

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Abstract

L'invention concerne des promoteurs qui induisent l'expression spécifique de racines des séquences nucléotidiques codantes qu'ils contrôlent; des cassettes d'expression, des vecteurs recombinants et des micro-organismes renfermant lesdits promoteurs; des plantes transgéniques transformées; ainsi qu'une technique permettant de les produire et une technique permettant d'isoler des promoteurs spécifiques de racines.
PCT/EP1999/008786 1998-11-16 1999-11-16 Promoteurs d'expression genique dans les racines de plantes WO2000029566A1 (fr)

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JP2000582550A JP2002530075A (ja) 1998-11-16 1999-11-16 植物の根における遺伝子発現のプロモーター
AU16518/00A AU1651800A (en) 1998-11-16 1999-11-16 Promoters for gene expression in the roots of plants
EP99959279A EP1135480A1 (fr) 1998-11-16 1999-11-16 Promoteurs d'expression genique dans les racines de plantes
CA002350186A CA2350186A1 (fr) 1998-11-16 1999-11-16 Promoteurs d'expression genique dans les racines de plantes

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Cited By (12)

* Cited by examiner, † Cited by third party
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US6907887B2 (en) 2001-06-08 2005-06-21 Vector Tobacco Ltd. Modifying nicotine and nitrosamine levels in tobacco
US6911541B2 (en) 2000-08-30 2005-06-28 North Carolina State University Promoter fragment that is recognized by the product of the tobacco Nic gene
US7189570B2 (en) 2000-11-07 2007-03-13 North Carolina State University Putrescine-n-methyltransferase promoter
WO2006066193A3 (fr) * 2004-12-16 2007-06-07 Ceres Inc Promoteurs actifs des racines et utilisations de ceux-ci
US7304220B2 (en) 1997-06-12 2007-12-04 North Carolina State University Regulation of quinolate phosphoribosyl transferase expression
WO2009104893A3 (fr) * 2008-02-19 2009-12-17 Industry Foundation Of Chonnam National University Promoteur d'expression spécifique à la racine provenant d'un gène aquaporin de capsicum annuum
KR100953763B1 (ko) * 2008-03-12 2010-04-21 대한민국 뿌리 특이 발현 프로모터ｍ
EP2213681A1 (fr) 2002-03-22 2010-08-04 Bayer BioScience N.V. Nouvelles protéines insecticides à base de Bacillus thuringiensis
WO2014142647A1 (fr) 2013-03-14 2014-09-18 Wageningen Universiteit Souches fongiques ayant une production améliorée d'acide citrique et d'acide itaconique
US10851383B2 (en) 2003-10-14 2020-12-01 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof
US11634723B2 (en) 2003-09-11 2023-04-25 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof
US11739340B2 (en) 2003-09-23 2023-08-29 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
DE102009023904B4 (de) 2009-06-04 2021-07-15 Bayerische Motoren Werke Aktiengesellschaft Kraftfahrzeug mit einem Kotflügel
JP5540068B2 (ja) 2010-02-17 2014-07-02 日本たばこ産業株式会社 植物内容成分の調節因子、およびその利用

Citations (1)

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WO1991013992A1 (fr) * 1990-03-16 1991-09-19 Advanced Technologies (Cambridge) Ltd. Promoteur pour plantes

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WO1991013992A1 (fr) * 1990-03-16 1991-09-19 Advanced Technologies (Cambridge) Ltd. Promoteur pour plantes

Non-Patent Citations (3)

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Title
BUCHER M ET AL: "Two genes encoding extension-like proteins are predominantly expressed in tomato root hair cells.", PLANT MOLECULAR BIOLOGY, (1997 NOV) 35 (4) 497-508., XP000882011 *
CONKLING M ET AL: "Isolation of transcriptionally regulated root-specific genes from tobacco", PLANT PHYSIOLOGY,US,AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, vol. 3, no. 93, 1 July 1990 (1990-07-01), pages 1203 - 1211, XP002080227, ISSN: 0032-0889 *
LAUTER F -R: "ROOT-SPECIFIC EXPRESSION OF THE LERSE-1 GENE IN TOMATO IS INDUCED BY EXPOSURE OF THE SHOOT TO LIGHT", MOLECULAR AND GENERAL GENETICS,DE,SPRINGER VERLAG, BERLIN, vol. 252, 1 January 1996 (1996-01-01), pages 751 - 754, XP002911926, ISSN: 0026-8925 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7425670B2 (en) 1997-06-12 2008-09-16 North Carolina State University Methods and compositions for protein production in tobacco plants with reduced nicotine
US7304220B2 (en) 1997-06-12 2007-12-04 North Carolina State University Regulation of quinolate phosphoribosyl transferase expression
US7605308B2 (en) 1997-06-12 2009-10-20 North Carolina State University Regulation of quinolate phosphoribosyl transferase expression
US7408098B2 (en) 1997-06-12 2008-08-05 North Carolina State University Regulation of quinolate phosphoribosyl transferase expression
US6911541B2 (en) 2000-08-30 2005-06-28 North Carolina State University Promoter fragment that is recognized by the product of the tobacco Nic gene
US7192771B2 (en) 2000-08-30 2007-03-20 North Carolina State University Plant promoter sequence
US7189570B2 (en) 2000-11-07 2007-03-13 North Carolina State University Putrescine-n-methyltransferase promoter
US6907887B2 (en) 2001-06-08 2005-06-21 Vector Tobacco Ltd. Modifying nicotine and nitrosamine levels in tobacco
EP2213681A1 (fr) 2002-03-22 2010-08-04 Bayer BioScience N.V. Nouvelles protéines insecticides à base de Bacillus thuringiensis
US11634723B2 (en) 2003-09-11 2023-04-25 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof
US11739340B2 (en) 2003-09-23 2023-08-29 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof
US10851383B2 (en) 2003-10-14 2020-12-01 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof
US7385105B2 (en) 2004-12-16 2008-06-10 Ceres, Inc. Root active promoters and uses thereof
WO2006066193A3 (fr) * 2004-12-16 2007-06-07 Ceres Inc Promoteurs actifs des racines et utilisations de ceux-ci
WO2009104893A3 (fr) * 2008-02-19 2009-12-17 Industry Foundation Of Chonnam National University Promoteur d'expression spécifique à la racine provenant d'un gène aquaporin de capsicum annuum
KR100974820B1 (ko) * 2008-02-19 2010-08-09 전남대학교산학협력단 고추 아쿠아포린 유전자 유래 뿌리 특이적 발현 프로모터및 이를 포함하는 뿌리 특이적 발현 벡터
KR100953763B1 (ko) * 2008-03-12 2010-04-21 대한민국 뿌리 특이 발현 프로모터ｍ
WO2014142647A1 (fr) 2013-03-14 2014-09-18 Wageningen Universiteit Souches fongiques ayant une production améliorée d'acide citrique et d'acide itaconique

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