WO2009095059A1 - Promoteurs de la biosynthèse du caoutchouc à partir de taraxacum koksaghyz et leur utilisation - Google Patents

Promoteurs de la biosynthèse du caoutchouc à partir de taraxacum koksaghyz et leur utilisation Download PDF

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WO2009095059A1
WO2009095059A1 PCT/EP2008/010563 EP2008010563W WO2009095059A1 WO 2009095059 A1 WO2009095059 A1 WO 2009095059A1 EP 2008010563 W EP2008010563 W EP 2008010563W WO 2009095059 A1 WO2009095059 A1 WO 2009095059A1
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nucleic acid
plant
acid sequence
seq
promoter
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Dirk PRÜFER
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Westfälische Wilhelms Universität Münster
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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
    • 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
    • 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

Definitions

  • the present invention relates to plant promoters and their uses.
  • the promoters may be used for expression of homologous or heterologous proteins in plants, in particular laticiferous plants, or for the expression of active nucleic acid molecules, such as sense and/or anti- sense RNA.
  • nucleic acid sequences having promoter activity as well as chimeric genes, vectors and recombinant (transgenic) cells and organism comprising these. Also provided are methods for making transgenic cells and organisms, especially plants and plant cells, comprising the promoters.
  • the promoters are in particular active with gene sequences that are associated with cis-prenyl transferase (rubber transferase) or gene sequences that are associated with small rubber particle protein, but can also drive the tissue specific expression of other proteins.
  • the promoters are useful for the production of various compounds in plants, such as for the improvement of the quantity and quality of rubber and for the production recombinant proteins for industrial and pharmaceutical applications and the like.
  • Rubber cis-1 ,4-poly isoprene
  • isoprenoid polymer with no known physiological function to the plant
  • Rubber is produced in about 2000 plant species with varying degrees of quality and quantity (Backhaus , 1985, Isr. J. Bot. 34 283-293). Rubber is limited to only a few plant families (See: Backhaus, Israel J. Bot., 34:283-293 (1985); and Archer et al., Chemistry and Physics of Rubber-like Substances, Bateman, L. ed., pp 41-72, MacLaren, London (1963)). Rubber is the raw material of choice for many commercial applications such as for heavy duty tires and other industrial uses requiring elasticity, flexibility and resilience.
  • Rubber is a polymer composed of between 320-35,000 isoprene molecules. These are linked by stepwise, head-to-tail, cis-1 ,4 condensations that form the polyisoprene chains. The stepwise isoprene additions are performed by a prenyltransferase enzyme [E. C.
  • RUT rubber transferase
  • rubber polymerase rubber polymerase
  • rubber cis 1-4 polyprenyl transferase 2.5.1.20] known as rubber transferase (RUT), rubber polymerase and rubber cis 1-4 polyprenyl transferase.
  • RUT rubber transferase
  • the exact structure of RUT has not yet been elucidated, it is thought that it concerns a membrane bound enzyme complex of which cis-prenyl transferase and small particle rubber protein form a part.
  • RuT has been ascribed as the sole enzyme responsible for rubber formation in plants (See: Backhaus, Israel J. Bot. 34: 283-293, (1985); Berndt, U.S. Government Res. Rep.
  • Laticiferous plants possess cells known as laticifers that contain latex, a colorless or milky sap that protects them against pathogens and insect herbivory.
  • Latex comprises a mixture of proteins, carbohydrates, oils and secondary metabolites, but most importantly it may contain rubber, which has diverse industrial uses. Although many plant species are known to produce natural rubber in their latex, the rubber tree Hevea brasiliensis is currently the only commercial source for natural latex. Malaysia, Thailand and Indonesia are today's major producers of natural rubber accounting for 80% of the world market.
  • brasiliensis clones in Asia and Africa lack genetic diversity are therefore highly susceptible to this disease, devastating losses are expected if SALB spreads to Asia.
  • Alternative sources of natural rubber is an important objective of plant research.
  • Alternative sources are also desirable for individuals with latex allergies.
  • T. koksaghyz (Taraxacum koksaghyz). T. koksaghyz was used intensively in North America and Europe as an alternative source for natural rubber during World War II, and although it produced rubber of excellent quality, it could not compete economically with the Heave rubber that became available again after WWII. The main reasons for non-economic production of rubber in T. koksaghyz was its poor agronomic performance. By contrast the common dandelion, T. officinale, which is a close relative of T. koksaghyz, has a much better agronomic performance, but does not produce rubber in significant quantities, although the cis 1-4 polyprenyl transferase and the small rubber particle protein genes are present
  • transgenic plant or plant cell or plant tissue or organ comprising a chimeric gene integrated in its genome, characterized in that said chimeric gene comprises a promoter operably linked to a homologous or heterologous nucleic acid sequence, wherein the promoter is selected from the group of:
  • nucleic acid sequence comprising at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2 (or its complement),;
  • nucleic acid sequence having promoter activity when introduced into plant cells, wherein said nucleic acid sequence comprising a sequence selected from:
  • nucleic acid sequence comprising at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2 (or its complement),;
  • Vectors, chimeric genes and host cells comprising the above sequences are also an embodiment of the invention. Further, a method is provided for making a transgenic plant or plant cell, comprising the steps of:
  • the method may further comprise growing the transgenic plant (or a derivative thereof, such as derived from crossing or selfing and wherein the derivative retains the chimeric gene) and harvesting the latex produced for further use (for instance for the generation and isolation of proteins or other compounds in the latex that result from transcription of the chimeric gene).
  • Such production of recombinant proteins whether pharmaceutically interesting, e.g. antibodies, insulin or industrial relevant proteins, is sometimes referred to as molecular farming / pharming
  • the use is not limited to pharmaceuticals, but could also include industrial recombinant proteins like enzymes.
  • the method may further comprise growing the transgenic plant (or a derivative thereof, such as derived from crossing or selfing and wherein the derivative retains the chimeric gene) and harvesting all or part of the plant, such as the leaves, fruit, seeds, etc. for further use.
  • the promoter sequences provided here exhibit a clear and specific expression of the operably linked gene, in this case the GUS gene from E. coli in Taraxacum koksaghyz. Additionally, it is shown here that the promoter sequence obtained from a Taraxacum koksaghyz is active in tobacco (Nicotiana benthamiana), thus the promoter is also capable of providing expression of genes in non-latex producing plants.
  • nucleic acid sequence refers to a DNA or RNA molecule in single or double stranded form, particularly a DNA having promoter activity according to the invention or a DNA encoding a protein or protein fragment.
  • isolated nucleic acid sequence refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.
  • protein or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3- dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a “protein”.
  • isolated protein is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
  • gene means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable transcription regulatory regions (e.g. a promoter).
  • a gene may thus comprise several operably linked sequences, such as a promoter, a 5' non-translated leader sequence (also referred to as 5'UTR 1 which corresponds to the transcribed mRNA sequence upstream of the translation start codon) comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3'non-translated sequence (also referred to as 3' untranslated region, or 3'UTR) comprising e.g. transcription termination sites and polyadenylation site (such as e.g. AAUAAA or variants thereof).
  • a promoter such as a promoter, a 5' non-translated leader sequence (also referred to as 5'UTR 1 which corresponds to the transcribed mRNA sequence upstream of the translation start codon) comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3'non-translated sequence (also
  • a “chimeric gene” refers to any gene, which is not normally found in nature in a species, in particular a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature.
  • the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region.
  • the term “chimeric gene” is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more sense sequences (e.g. coding sequences) or to an antisense (reverse complement of the sense strand) or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription).
  • a "3' UTR” or “3' non-translated sequence” refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA or variants thereof).
  • a polyadenylation signal such as e.g. AAUAAA or variants thereof.
  • the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).
  • “Expression of a gene” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into a RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment) or which is active itself (e.g. in posttranscriptional gene silencing or RNAi, or silencing through miRNAs).
  • An active protein in certain embodiments refers to a protein having a dominant-negative function due to a repressor domain being present.
  • the coding sequence is preferably in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment.
  • the DNA sequence is preferably present in the form of an antisense DNA or an inverted repeat DNA 1 comprising a short sequence of the target gene in antisense or in sense and antisense orientation.
  • Down-regulation of gene expression can also take place through the action of microRNAs (miRNA), endogenous 21-24 nucleotide small RNAs processed from stem-loop RNA precursors (pre-miRNAs), Incorporated into a RNA- induced silencing complex (RISC), miRNAs down-regulate gene expression by mRNA cleavage or translational repression.
  • miRNA microRNAs
  • pre-miRNAs endogenous 21-24 nucleotide small RNAs processed from stem-loop RNA precursors
  • RISC RNA- induced silencing complex
  • Ectopic expression refers to expression in a tissue in which the gene is normally not expressed.
  • a “transcription regulatory sequence” is herein defined as a nucleic acid sequence that is capable of regulating the rate of transcription of a nucleic acid sequence operably linked to the transcription regulatory sequence.
  • a transcription regulatory sequence as herein defined will thus comprise all of the sequence elements necessary for initiation of transcription (promoter elements), for maintaining and for regulating transcription, including e.g. attenuators or enhancers, but also silencers.
  • promoter elements e.g. attenuators or enhancers, but also silencers.
  • regulatory sequences found downstream (3') of a coding sequence are also encompassed by this definition.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream (5 1 ) with respect to the direction of transcription of the transcription initiation site of the gene (the transcription start is referred to as position +1 of the sequence and any upstream nucleotides relative thereto are referred to using negative numbers), and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA domains (cis acting sequences), including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • Examples of eukaryotic cis acting sequences upstream of the transcription start (+1) include the TATA box (commonly at approximately position -20 to -30 of the transcription start), the CAAT box (commonly at approximately position -75 relative to the transcription start), 5'enhancer or silencer elements, etc.
  • a "constitutive" promoter is a promoter that is active in essentially all tissues and organs under most physiological and/or developmental conditions. More preferably, a constitutive promoter is active under essentially all physiological and developmental conditions in all major organs, such as at least the leaves, stems, roots, seeds, fruits and flowers. Most preferably, the promoter is active in all organs under most (preferably all) physiological and developmental conditions.
  • tissue-specific or tissue-preferred promoter can also be referred to as being “constitutively active”.
  • the promoter is thus active under most developmental and/or physiological conditions, albeit in only a specific tissue or mainly in a specific tissue.
  • a "promoter which has constitutive activity” or which is “constitutive” in a plant or plant cell refers, therefore, to a nucleic acid sequence which confers transcription in the plant or plant cells in the specific tissue under most physiological and developmental conditions.
  • an “inducible” promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmentally regulated.
  • tissue specific promoter is only active in specific types of tissues or cells, such as latex producing cells.
  • the promoter activity can therefore be described by referring to the circumstances under which the promoter confers transcription of the nucleic acid sequence operably linked downstream (3') of the promoter.
  • tissue preferred promoter is preferentially, but not exclusively, active in certain tissues or cells, such as for example in latifers, cells that produce latex.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter, or a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame so as to produce a "chimeric protein".
  • a “chimeric protein” or “hybrid protein” is a protein composed of various protein "domains” (or motifs) which is not found as such in nature but which are joined to form a functional protein, which displays the functionality of the joined domains (for example a DNA binding domain or a repression of function domain leading to a dominant negative function).
  • a chimeric protein may also be a fusion protein of two or more proteins occurring in nature.
  • domain as used herein means any part(s) or domain(s) of the protein with a specific structure or function that can be transferred to another protein for providing a new hybrid protein with at least the functional characteristic of the domain.
  • target peptide refers to amino acid sequences which target a protein to intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or to the extracellular space (secretion signal peptide).
  • a nucleic acid sequence encoding a target peptide may be fused (in frame) to the nucleic acid sequence encoding the amino terminal end (N-terminal end) of the protein.
  • a "nucleic acid construct” or “vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell.
  • the vector backbone may for example be a binary or superbinary vector (see e.g. US 5,591 ,616, US2002138879 and WO 95/06722), a co- integrate vector or a T-DNA vector, as known in the art and as described elsewhere herein, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence / promoter is already present, only a desired nucleic acid sequence (e.g.
  • Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like (see below).
  • a "host cell” or a “recombinant host cell” or “transformed cell” are terms referring to a new individual cell (or organism), arising as a result of the introduction into said cell of at least one nucleic acid molecule, especially comprising a chimeric gene encoding a desired protein or a nucleic acid sequence which upon transcription yields an antisense RNA or an inverted repeat RNA (or hairpin RNA) for silencing of a target gene/gene family.
  • the host cell is preferably a plant cell, but may also be a bacterial cell, a fungal cell (including a yeast cell), etc.
  • the host cell may contain the nucleic acid construct as an extra-chromosomally (episomal) replicating molecule, or more preferably, comprises the chimeric gene integrated in the nuclear or plastid genome of the host cell.
  • selectable marker is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker.
  • Selectable marker gene products confer, for example, antibiotic resistance, or more preferably, herbicide resistance or another selectable trait such as a phenotypic trait (e.g. a change in pigmentation) or a nutritional requirement.
  • reporter is mainly used to refer to visible markers, such as green fluorescent protein (GFP), eGFP, luciferase, GUS and the like, as well as nptll markers and the like.
  • ortholog of a gene or protein refers herein to the homologous gene or protein found in another species, which has the same function as the gene or protein, but (usually) diverged in sequence from the time point on when the species harbouring the genes diverged (i.e. the genes evolved from a common ancestor by speciation). Orthologs of a gene from one plant species may thus be identified in other plant species based on both sequence comparisons (e.g. based on percentages sequence identity over the entire sequence or over specific domains) and functional analysis.
  • homologous and heterologous refer to the relationship between a nucleic acid or amino acid sequence and its host cell or organism, especially in the context of transgenic organisms.
  • a homologous sequence is thus naturally found in the host species (e.g. a tomato plant transformed with a tomato gene), while a heterologous sequence is not naturally found in the host cell (e.g. a tomato plant transformed with a sequence from potato plants).
  • a heterologous sequence is not naturally found in the host cell (e.g. a tomato plant transformed with a sequence from potato plants).
  • the term "homolog” or “homologous” may alternatively refer to sequences which are descendent from a common ancestral sequence (e.g. they may be orthologs).
  • Stringent hybridisation conditions can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence.
  • the stringency of the hybridization conditions are sequence dependent and will be different in different circumstances.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequences at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe.
  • stringent conditions will be chosen in which the salt (NaCI) concentration is about 0.02 molar at pH 7 and the temperature is at least 60 0 C. Lowering the salt concentration and/or increasing the temperature increases stringency.
  • Stringent conditions for RNA-DNA hybridisations are for example those which include at least one wash in 0.2X SSC at 63°C for 20 min, or equivalent conditions.
  • Stringent conditions for DNA-DNA hybridisation are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50 0 C, usually about 55 0 C, for 20 min, or equivalent conditions. See also Sambrook et a/. (1989) and Sambrook and Russell (2001).
  • High stringency conditions can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6x SSC (2Ox SSC contains 3.0 M NaCI, 0.3 M Na-citrate, pH 7.0), 5x Denhardt's (100X Denhardt's contains 2% Ficoll, 2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium dodecyl sulphate (SDS), and 20 ⁇ g/ml denaturated carrier DNA (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) as non-specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0.1 * SSC, 0.1% SDS.
  • 6x SSC contains 3.0 M NaCI, 0.3 M Na-citrate, pH 7.0
  • 5x Denhardt's 100X Denhardt's contains 2% Ficol
  • Mode stringency refers to conditions equivalent to hybridization in the above described solution but at about 60-62° C. In that case the final wash is performed at the hybridization temperature in 1x SSC, 0.1% SDS.
  • Low stringency refers to conditions equivalent to hybridization in the above described solution at about 50-52° C. In that case, the final wash is performed at the hybridization temperature in 2x SSC, 0.1% SDS. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
  • Sequence identity and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as “substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below).
  • a global alignment algorithms e.g. Needleman Wunsch
  • GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths.
  • the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).
  • Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program "needle” (using the global Needleman Wunsch algorithm) or "water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for 'needle' and for 'water' and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFuII for DNA). When sequences have a substantially different overall lengths, local alignments, such as using the Smith Waterman algorithm, are preferred. Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc.
  • plant or “plants” (or a plurality of plants) according to the invention is made, it is understood that also plant parts (cells, tissues or organs, seeds, severed or harvested parts, leaves, seedlings, flowers, pollen, fruit, stems, roots, callus, protoplasts, etc), progeny or clonal propagations of the plants which retain the distinguishing characteristics of the parents (e.g. presence of a trans-gene), such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived therefrom are encompassed herein, unless otherwise indicated.
  • plant promoters which exhibit an activity in plants and in particular in latex producing plants. Such promoters are desired for the controlled expression of nucleic acid sequences in transgenic plants.
  • promoters for the cpp and srpp genes have entirely different non-rubber biosynthesis applications, namely the laticifer-specific expression of recombinant proteins that have industrial or pharmaceutical applications.
  • Latex of Taraxacum officinale contains only a limited number of proteins and therefore latex expressed recombinant proteins will be relatively easy to extract and purify.
  • latex and laticifers are a favourable environment for the stable expression and accumulation of proteins. This may be especially useful in combination with the reduction of the activity of the latex specific poly phenol oxidases, which will reduce the coagulation of the latex and facilitate the extraction.
  • Taraxacum plants can be grown hyponically in hydroculture in a greenhouse giving easy access to the roots which contain the highest latex concentrations. Roots could be partially cut and the latex with the recombinant proteins could be easily harvested. A high concentration of the recombinant protein compared to other proteins in the latex will make the isolation of the recombinant protein economically feasible.
  • Another benefit of the expression of the recombinant protein in the laticifers is that high concentrations of the recombinant protein are unlikely to be toxic for the plant itself. This is because the latificers have a non-vital function, in contrast to the other part of the vascular tissues. High concentrations of recombinant protein typically do not affect the growth of the plants negatively, adding to the advantages of the present invention.
  • a method to increase both the agronomic performance and the rubber production is to place the native T.officinale cis 1-4 polyprenyl transferase and the small rubber particle proteins under the T. koksaghyz promoters of these genes, in line with the present invention.
  • the promoters of the present invention can also be advantageously used in methods to increase the production of proteins in other laticifers such as those listed herein elsewhere.
  • Taraxacum roots contain a high percentage of inulin, a polyfructan which is an ideal precursor for the second generation biofuels (2,5-dimethylfuran, DMF) and plastics (5-hydroxymethylfurfural, HMF).
  • the promoters of the present invention can be used to improve Taraxacum as a valuable dual purpose inulin-rubber crop for the temperate regions.
  • the koksaghyz cpt and srpp promoters may also be useful in other rubber producing crops like guayule and Hevea if the koksaghyz promoters give a higher rubber production than the native promoters of these species
  • the invention provides promoter regions of Taraxacum genes (Taraxacum cis-prenyltransferase and small rubber particle protein and their orthologs and homologs thereof) which confer expression in host plants, such as other latex producing plants or cultivated plants such as tobacco (Nicotiana benthamania), tomato (Solarium lycopersicum), other Solanaceae and other plant families and species, and in particular confer expression in the laticifers and also preferably in the vascular system.
  • Taraxacum genes Teraxacum cis-prenyltransferase and small rubber particle protein and their orthologs and homologs thereof
  • host plants such as other latex producing plants or cultivated plants such as tobacco (Nicotiana benthamania), tomato (Solarium lycopersicum), other Solanaceae and other plant families and species, and in particular confer expression in the laticifers and also preferably in the vascular system.
  • isolated nucleic acid sequences (preferably genomic or synthetic DNA sequences), having promoter activity in plant cells, are provided which show strong, specific activity in laticeferous plants, and in particular in the laticifers and/or in the vascular system.
  • a specific promoter comprising or consisting of SEQ ID NO: 1 or SEQ ID NO: 2 or a nucleotide sequence essentially similar thereto (referred to as "variants”, see definition below), or active (functional) fragments of any of these which have promoter activity in plants, preferably one or more lactiferous plants , such as fragments of at least 200, 300, 400, 500, 600, 800, 900, 1000, 1200, 1500, 2000, 2400 or more consecutive nucleotides of SEQ ID NO: 1 , or 2, or of variants thereof.
  • active fragments or “functional fragments”, or “fragments having promoter activity” refer to nucleic acid fragments which are capable of conferring transcription in one or more cells found in one or more different types of plant tissues and organs (e.g. on stems, leaves, flower buds or flower parts).
  • active fragments Preferably have at least a similar strength (or higher strength) as the promoter of SEQ ID NO: 1 or 2 . This can be tested as described below, by transforming a plant with such a fragment, preferably operably linked to a reporter gene, and assaying the promoter activity qualitatively (spatio-temporal transcription) and/or preferably quantitatively.
  • DNA fragments may be generated in a number of ways, e.g. using de novo DNA synthesis, or restriction enzymes, or terminal nucleases, etc. Deletion analysis, whereby fragments are generated which comprise 5' deletions of various sizes can for example be used to create stronger and/or more specific transcriptional activity.
  • variants of the above specific promoters, and functional fragments of such variants.
  • These variants include nucleic acid sequences essentially similar to SEQ ID NO: 1 and/or SEQ ID NO: 2 (and functional fragments of these variant sequences, as described above), and which have promoter activity, i.e. which are also capable of providing (preferably constitutive) transcription in plants, in particular lactiferous plants.
  • Sequences which are "essentially similar" to SEQ ID NO: 1 and/or 2 are nucleic acid sequences comprising at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more nucleic acid sequence identity to SEQ ID NO: 1 and/or to SEQ ID NO: 2, using the Needleman and Wunsch or the Smith Waterman Pairwise alignment (e.g. program “needle” or “water” in Embosswin, e.g. version 2.10.0, with default gap creation and gap extension penalties) and which are specific in their activity.
  • the activity of the variants (and functional fragments) is strong in lactiferous and other plants, i.e.
  • the cell type specificity is at least as the specificity of SEQ ID NO: 1 or 2 or more specific.
  • the activity of these variants (and functional fragments thereof) is insensitive to one or more biotic and/or abiotic stresses.
  • nucleic acid hybridization can be used to identify DNA sequences in other plant species or varieties which hybridize to SEQ ID NO: 1 or 2, or to fragments of these, under stringent or moderately stringent hybridization conditions.
  • sequence databases can be screened in silico for variant sequences using known algorithms, such as BLAST, FASTA, etc.
  • BLAST Altschul et al.
  • FASTA Altschul et al.
  • the cDNA libraries may be screened for cisprenyltransferase or small rubber particle protein cDNAs (using e.g. probes or primers derived from SEQ ID NO: 1 or 2, or fragments or variants thereof). Equally, differential display methods (such as cDNA-AFLP) may be used to identify such transcripts. Methods such as TAIL-PCR (Liu et al. 1995, Genomics 25(3):674-81 ; Liu et al. 2005, Methods MoI Biol. 286:341-8), Linker-PCR, or Inverse PCR (IPCR) may be used to isolate the upstream transcription regulatory region of the gene.
  • TAIL-PCR Liu et al. 1995, Genomics 25(3):674-81 ; Liu et al. 2005, Methods MoI Biol. 286:341-8
  • Linker-PCR or Inverse PCR (IPCR) may be used to isolate the upstream transcription regulatory region of the gene.
  • Variants of the same genes i.e. orthologs and/or homologs of cisprenyltransferase and small rubber particle protein include for example nucleic acid sequences (DNA or RNA) or amino acid sequences comprising at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more nucleic acid or amino acid sequence identity to the nucleotide sequence or amino acid sequence of known cisprenyltransferase and small rubber particle protein such as disclosed inter alia in Genbank and in US WO01/21650 and WO2004/044173. Sequence identity is determined by pairwise alignment using the Needleman and Wunsch or the Smith Waterman Pairwise alignment (e.g.
  • the promoters of these variants are preferably also constitutive and/or specific in their activity. Methods such as cDNA-AFLP, other PCR based methods or Northern hybridization may be used to isolate or identify such genes. Their promoter can be cloned using known methods.
  • the promoter is obtained from a cisprenyltransferase and small rubber particle protein gene from a plant belonging to the genus Taraxacum, such as species of the species koksaghyz, and genera like Lactuca, Nicotiana, Solarium, Capsicum, Petunia, Coffea, etc. Especially orthologs from wild species are desired.
  • Whether a nucleic acid sequence (or fragment of variant) has constitutive promoter activity, i.e. is capable of conferring transcription specifically in the latex producing system so the plant, whether the activity is "strong”, can be determined using various methods. Generally, one can distinguish qualitative methods and quantitative methods. Qualitative methods (such as histological GUS staining) are used to determine the spatio-temporal activity of the promoter (is the promoter active or not in a certain tissue or organ, or under certain environmental/developmental conditions), while quantitative methods (such as fluorometric GUS assays) also quantify the level of activity, compared to controls. Suitable controls are, for example, plants transformed with empty vectors (negative control) or transformed with constructs comprising other promoters.
  • a cloned or synthetic nucleic acid molecule such as SEQ ID NO: 1 or 2 , or variants thereof, or fragments of any of these, may be operably linked to a known nucleic acid sequence (e.g. a reporter gene, such as gusA, or any gene encoding a specific protein) and may be used to transform a plant cell using known methods and regenerate a plant therefrom.
  • a known nucleic acid sequence e.g. a reporter gene, such as gusA, or any gene encoding a specific protein
  • the activity of the promoter can, for example, be assayed (and optionally quantified) by detecting the level of RNA transcripts of the downstream nucleic acid sequence, especially in the latex producing cells. This may be done using quantitative methods, such as e.g. quantitative RT-PCR or other PCR based methods, and the like.
  • the reporter protein or the activity of the reporter protein may be assayed and quantified.
  • the reporter gene is the GUS gene
  • a fluorometric GUS assay may be used, as described in the Examples.
  • the quantitative promoter activity levels of transformed plants or plant cells maintained under normal physiological (non-stress) conditions can be compared to levels of plants or plant cells which are exposed to one or more biotic or abiotic stresses.
  • relative or absolute activity levels in the latex producing cells can be compared to constitutive control promoters, such as the 35S promoter, double-35S promoter, or to other promoters which have activity in latex producing cells. It is understood that preferably average promoter activity levels are determined and compared using statistical methods.
  • RNA transcript or reporter protein or its activity.
  • One simple test employs for example histochemical GUS staining, whereby visual assessment of blue colour indicates activity in latex producing cells and at various developmental stages of the latex producing cells.
  • the promoter activity is constitutive and preferably also strong in latex producing cells, especially in the host species or variety into which the sequence is introduced.
  • Constitutive activity means that the transcript of any nucleic acid sequence operably linked to the promoter is preferably produced in latex producing cells under most (normal, non-stressed) physiological and developmental conditions.
  • the promoters according to the invention are preferably not active in epidermal cells.
  • the promoters are active in all latex producing cells found in roots, stems, flowers and/or (young) leaves.
  • roots are harvested to isolate the latex.
  • the promoters according to the invention provide strong, constitutive activity in cells of all plant species, both dicotyledonous species and monocotyledonous species, such as described below (e.g. tomato, tobacco, Brassica, melon and lettuce and others).
  • plants that have laticifers in which plants, and preferably in the laticifers, genes are expressed using the promoters of the present invention that contain a vascular system that can be tapped, wherein, in a preferred embodiment, the latex contains a high concentration of rubber or protein or both and wherein preferably the protein is easy to isolate
  • the strength (quantitative activity) of the promoters according to the invention can be determined quantitatively using various known methods. For example, the amount of transcribed transcript (mRNA) can be quantified using quantitative RT-PCR or northern blotting.
  • the promoter strength is at least essentially equal to the activity in the latex producing cells of the Taraxacum koksaghyz under normal (non- stressed) conditions. "Strong" means, thus, that the promoter strength is preferably at least about identical, but more preferably stronger than the activity in the latex producing cells of the Taraxacum koksaghyz under normal, non-stressed conditions.
  • the average quantitative promoter activity in the transgenic plants comprising the promoter is at least equivalent to the activity in the latex producing cells of the Taraxacum koksaghyz, or is at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, or more, higher than the average activity in the latex producing cells of the Taraxacum koksaghyz. It is understood that the same copy number and zygosity level of transformants should be compared, e.g. hemizygous or homozygous for the transgene. Preferably, single copy transformants are identified and compared.
  • "strong" may mean that the promoter strength is preferably at least about identical, but more preferably stronger than that of at least one of the promoters consisting of SEQ ID NO: 1 or 2 in plants under normal, non-stressed conditions.
  • the average quantitative promoter activity in the transgenic plants is at least equivalent to the activity of at least one of the promoters consisting of SEQ ID NO: 1 or 2, or is at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, or more, higher than the average activity of at least one of the promoters consisting of SEQ ID NO: 1 or 2 in the in the latex producing cells of the Taraxacum koksaghyz. It is understood that the same copy number and zygosity level of transformants should be compared, e.g. hemizygous or homozygous for the transgene. Preferably, single copy transformants are identified and compared.
  • any of the above promoters for the expression of homologous or heterologous nucleic acid sequences in a recombinant cell or organism, especially a plant cell or plant is provided.
  • This use comprises operably linking the promoter to a homologous or heterologous nucleic acid sequence and transforming a plant or plant cell, as described further below.
  • the focus above is on the use of the promoters according to the invention in plants and plant cells, in particular laticifers, it is also an embodiment of the invention to use the promoters for the expression of homologous or heterologous nucleic acid sequences in other cells and organisms, such as in any prokaryotic or eukaryotic cells or organisms, e.g. bacteria, fungi (including yeasts, such as Pichia, Hansenula, etc.), mammals, human cells or cell lines, etc.
  • prokaryotic or eukaryotic cells or organisms e.g. bacteria, fungi (including yeasts, such as Pichia, Hansenula, etc.), mammals, human cells or cell lines, etc.
  • any of the above nucleic acid sequences having promoter activity are used to make chimeric genes, and vectors comprising these for transfer of the chimeric gene into a host cell and expression of an operably linked homologous or heterologous nucleic acid sequence in host cells, such as cells, tissues, organs or whole organisms derived from transformed cell(s).
  • Host cells are preferably plant cells. Any plant may be a suitable host, such as monocotyledonous plants or dicotyledonous plants, for example maize/corn (Zea species, e.g. Z. mays, Z. diploperennis (chapule), Zea luxurians (Guatemalan teosinte), Zea mays subsp. huehuetenangensis (San Antonio Huista teosinte), Z. mays subsp. mexicana (Mexican teosinte), Z. mays subsp. parviglumis (Balsas teosinte), Z. perennis (perennial teosinte) and Z.
  • Zea species e.g. Z. mays, Z. diploperennis (chapule), Zea luxurians (Guatemalan teosinte), Zea mays subsp. huehuetenangensis (S
  • ramosa wheat (Triticum species), barley (e.g. Hordeum vulgare), oat (e.g. Avena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypium species, e.g. G. hirsutum, G. barbadense), Brassica spp. (e.g. B. napus, B. juncea, B. oleracea, B.
  • rapa, etc. sunflower (Helianthus annus), tobacco (Nicotiana species), alfalfa (Medicago sativa), rice (Oryza species, e.g. O. sativa indica cultivar-group or japonica cultivar-group), forage grasses, pearl millet (Pennisetum species, e.g. P. glaucum), tree species, vegetable species, such as Lycopersicon ssp (recently reclassified as belonging to the genus Solanum), e.g. tomato (L. esculentum, syn. Solanum lycopersicum) such as e.g. cherry tomato, var. cerasiforme or current tomato, var.
  • pimpinellifolium or tree tomato (S. betaceum, syn. Cyphomandra betaceae), potato (Solanum tuberosum) and other Solanum species, such as eggplant (Solanum melongena), pepino (S. muricatum), cocona (S. sessiliflorum) and naranjilla (S. quitoense); peppers (Capsicum annuum, Capsicum frutescens), pea (e.g. Pisum sativum), bean (e.g.
  • Phaseolus species Phaseolus species), carrot (Daucus carota), Lactuca species (such as Lactuca sativa, Lactuca indica, Lactuca perennis), cucumber (Cucumis sativus), melon (Cucumis melo), zucchini (Cucurbita pepo), squash (Cucurbita maxima, Cucurbita pepo, Cucurbita mixta), pumpkin (Cucurbita pepo), watermelon (Citrullus lanatus syn. Citrullus vulgaris), fleshy fruit species (grapes, peaches, plums, strawberry, mango, melon), ornamental species (e.g.
  • Rose Petunia, Chrysanthemum, Lily, Tulip, Gerbera species
  • woody trees e.g. species of Populus, Salix, Quercus, Eucalyptus
  • fibre species e.g. flax (Linum usitatissimum) and hemp (Cannabis sativa)
  • species of the following genera may be transformed: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Cucumis, Hyoscyamus, Lycopersicon, Solanum, Nicotiana, Malus, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Citrullus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Browaalia, Glycine, Pisum, Phaseolus, Gossypium, Glycine, Lolium, Festuca, A
  • a further preference is for each of Cucurbita, Brassica, Lycopersicon, Solanum, Oryza and Zea.
  • a preference is for each of Avena, Medicago, Capsicum, Nicotiana, Lactuca, Pisum, Cucumis, Cucurbita, Brassica, Solanum (including Lycopersicon), Oryza and Zea.
  • the laticiferous plants or parts thereof are selected from the group of plant families consisting of Asteraceae, Euphorbiaceae and Moraceae, wherein selection from the group consisting of Agoseris glauca, Apios americana, Apocynum androsaemifolium, Apocynum cannabinum, Asclepias asperula, Asclepias brachystephana, Asclepias californica, Asclepias decumbens, Asclepias eriocarpa, Asclepias erosa, Asclepias galioides, Asclepias hallii, Asclepias incarnata, Asclepias involucrata, Asclepias lanceolata, Asclepias latifolia, Asclepias mexicana, Asclepias valifoli
  • Asclepias rubra Asclepias speciosa, Asclepias subulata, Asclepias sullivantii, Asclepias syriaca, Asclepias tuberosa, Asclepias viridiflora, Chrysothamnus graveolens, Chrysothamnus nauseosus, Chrysothamnus viscidiflorus, Cynanchum acutum, Eucommia ulmoides, Euonymus europaeus, Euonymus hamiltonianus, Euonymus hamiltonianus maackii, Euonymus hamiltonianus sieboldianus, Euonymus japonicus, Euonymus latifolius, Euonymus verrucosus, Euphorbia lathyris, Hevea brasiliensis, Hevea brasiliensis, Hymenoxys richardsonii, Jatroph
  • said laticiferous plants or parts thereof are selected from the group consisting of Hevea brasiliensis, Parthenium argentatum, Sonchus asper, Chrysothamnus nauseosus, Taraxacum koksaghyz, and Taraxacum officinale.
  • chimeric genes and vectors for introduction of chimeric genes into the genome of host cells, is generally known in the art.
  • the promoter sequence is operably linked to another nucleic acid sequence which is to be transcribed in the host cells, using standard molecular biology techniques.
  • the promoter sequence may already be present in a vector so that the nucleic acid sequence which is to be transcribed is simply inserted into the vector downstream of the promoter sequence.
  • the vector is then used to transform the host cells and the chimeric gene is preferably inserted in the nuclear genome or into the plastid, mitochondrial or chloroplast genome, so that the downstream nucleic acid sequence is expressed due to the activity of the promoter (e. g., Mc Bride et al., 1995 Bio/Technology 13, 362; US 5,693, 507).
  • a chimeric gene therefore, preferably comprises a promoter as described above, operably linked to a homologous or heterologous nucleic acid sequence, and optionally followed by a 3' nontranslated nucleic acid sequence (3 1 UTR).
  • the homologous or heterologous nucleic acid sequence may be a sequence encoding a protein or peptide, or it may be a sequence which is transcribed into an active RNA molecule, such as an sense and/or antisense RNA (sense and antisense RNA includes for example dsRNA or stem-loop RNA structures) suitable for silencing a gene or gene family in the host cell or organism.
  • the (specific) promoter-comprising chimeric gene can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a transformed plant that has an altered phenotype due to the expression of the chimeric gene.
  • a T-DNA vector comprising the promoter (or variant or fragment as described above) operably linked to a further nucleic acid sequence, in Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0 116 718, EP 0 270 822, PCT publication WO 84/02913 and published European Patent application EP 0 242 246 and in Gould et al. (1991 , Plant Physiol. 95,426-434).
  • the construction of a T-DNA vector for Agrobacterium mediated plant transformation is well known in the art.
  • the T-DNA vector may be either a binary vector as described in EP 0 120 561 and EP 0 120 515 or a co-integrate vector which can integrate into the Agrobacterium Ti- plasmid by homologous recombination, as described in EP 0 116 718.
  • T-DNA vectors each contain the promoter operably linked to the nucleic acid sequence to be transcribed between T-DNA border sequences, or at least located to the left of the right border sequence. Border sequences are described in Gielen et al. (1984, EMBO J 3,835-845). Of course, other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 223 247, or particle or microprojectile bombardment as described in US 2005/055740 and WO
  • transformation of the plastid genome preferably the chloroplast genome
  • transformation of the plastid genome is included in the invention.
  • plastid genome transformation is that the risk of spread of the transgene(s) can be reduced.
  • Plastid genome transformation can be carried out as known in the art, see e.g. Sidorov VA et al. 1999, Plant J.19: 209-216 or Lutz KA et al. 2004, Plant J. 37(6):906-13.
  • the resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants containing the transgene.
  • Single copy transformants can be selected, using e.g. Southern Blot analysis or PCR based methods or the Invader® Technology assay (Third Wave Technologies, Inc.).
  • Transformed cells and plants can easily be distinguished from non-transformed ones by the presence of the chimeric gene.
  • the sequences of the plant DNA flanking the insertion site of the transgene can also be sequenced, whereby an "Event specific" detection method can be developed, for routine use. See for example WO 01/41558, which describes elite event detection kits (such as PCR detection kits) based for example on the integrated sequence and the flanking (genomic) sequence.
  • the nucleic acid sequence which is to be transcribed, and optionally translated (if it is a coding sequence), is inserted into the plant genome so that the sequence to be transcribed is upstream (i.e. 5') of suitable 3'end transcription regulation signals ("3'end”) (i.e. transcript formation and polyadenylation signals).
  • 3'end transcription regulation signals
  • Polyadenylation and transcript formation signals include those of the nopaline synthase gene ("3' nos") (Depicker et al., 1982 J. Molec. Appl.
  • the nucleic acid sequence to be expressed is in one embodiment a sequence encoding a protein or peptide, including hybrid proteins or peptides or fusion proteins.
  • the coding sequence may be of any origin, i.e. plant, fungus (including yeast), animal, bacterial, synthetic, viral, human, etc. It may also comprise a sequence encoding a targeting peptide, such as a secretion signal peptide or a plastid targeting signal.
  • a coding sequence may also be linked in-frame to a gene encoding a selectable or scorable marker, such as for example the neo (or nptll) gene (EP 0 242 236) conferring kanamycin resistance, so that the cell expresses a fusion protein which is easily detectable.
  • a selectable or scorable marker such as for example the neo (or nptll) gene (EP 0 242 236) conferring kanamycin resistance, so that the cell expresses a fusion protein which is easily detectable.
  • a selectable or scorable marker such as for example the neo (or nptll) gene (EP 0 242 236) conferring kanamycin resistance, so that the cell expresses a fusion protein which is easily detectable.
  • the coding region cDNA or genomic DNA
  • examples of the coding regions of the following genes are preferably operably linked to a promoter according to the invention:
  • secondary metabolite biosynthesis genes or pathways including genes for the production of therapeutic and/or pharmacologically and cosmetically important products or industrially valuable compounds, genes providing nutritional or nutraceutical compounds, flavourants or scents, aromas .
  • the chimeric genes or vectors according to the invention can also be used to transform microorganisms, such as bacteria (e.g. Escherichia coli, Pseudomonas, Agrobacterium, Bacillus, etc.) or fungi or algae or insects, or the genes or vectors may be used to engineer viruses.
  • Transformation of bacteria with nucleic acid sequence of this invention, incorporated in a suitable cloning vehicle, can be carried out in a conventional manner, preferably using conventional electroporation techniques as described in Maillon et al. (1989, FEMS Microbiol. Letters 60, 205) and WO 90/06999.
  • codon usage of the nucleic acid sequence may be optimized accordingly (likewise, for expression of coding sequences in plant cells, codon usage of the nucleic acid sequence may be optimized as known), lntron sequences should be removed and other adaptations for optimal expression may be made as known.
  • transgenic plants comprising the promoter of the invention, operably linked to a protein or polypeptide encoding nucleic acid sequence, as described further below.
  • the promoters according to the invention are used to make a chimeric gene and vector for gene silencing, whereby the promoter is operably linked to a sense and/or antisense nucleic acid sequence of a target gene (endogenous gene or gene family which is to be silenced specifically in latex producing cells).
  • Gene silencing refers to the down-regulation or complete inhibition of gene expression of one or more target genes.
  • the use of inhibitory RNA to reduce or abolish gene expression is well established in the art and is the subject of several reviews (e.g. Baulcombe, 1996, Plant Cell 8: 1833-1844; Stam et al., 1997, Plant Journal 12: 63-82; Depicker and Van Montagu, 1997, Curr. Opinion Cell Biol. 9: 373-382).
  • technologies available to achieve gene silencing in plants such as chimeric genes which produce antisense RNA of all or part of the target gene (see e.g. EP 0 140 308 B1 , EP 0 240 208 B1 and EP 0 223 399 B1), or which produce sense RNA (also referred to as co-suppression), see EP 0 465 572 B1.
  • a vector according to the invention may therefore comprise a promoter accrdoing to the invention operably linked to a sense and/or antisense DNA fragment of a target gene.
  • Short (sense and antisense) stretches of the target gene sequence such as at least about 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides of coding or non-coding sequence may be sufficient. Longer sequences are frequently also used, such as at least about 100, 200, 250, 300, 400, 500, 1000, 1500 nucleotides, or more.
  • the sense and antisense fragments are separated by a spacer sequence, such as an intron, which forms a loop (or hairpin) upon dsRNA formation.
  • Any stretch of the target gene may be used to make a gene silencing vector and a transgenic plant in which the target gene or gene family is silenced.
  • a convenient way of generating hairpin constructs is to use generic vectors such as pHANNIBAL and pHELLSGATE, vectors based on the Gateway® technology (see Wesley et al. 2004, Methods MoI Biol. 265:117-30; Wesley et al. 2003, Methods MoI Biol. 236:273-86 and Helliwell & Waterhouse 2003, Methods 30(4):289-95.), all incorporated herein by reference.
  • the family members in a host plant can be silenced.
  • transgenic plants comprising the promoter according to the invention, operably linked to a sense and/or antisense DNA fragment of a target gene nucleic acid sequence and exhibiting a target gene silencing phenotype.
  • the phenotype will depend on the function of the gene, and may be a chemical or molecular change, macroscopically visible or not visible.
  • Such chimeric genes and vectors can, therefore, also be used to determine or verify the function of genes in lactiferous plants.
  • the chimeric genes according to the invention may be introduced stably into the host genome or may be present as an episomal unit.
  • Transgenic cells and organisms are provided, obtainable by the above methods. These cells and organisms are characterized by the presence of a chimeric gene in their cells or genome by the presence of a promoter according to the invention.
  • the mRNA transcript or the translated protein may alter the phenotype of the cells or organism.
  • the chimeric gene introduced into the plant is composed of parts which all occur naturally in the host genus or species, e.g.
  • the promoter according to the invention from the genus Taraxacum is used and operably linked to a nucleic acid sequence also from the genus Taraxacum and optionally a 3 1 UTR from the genus Taraxacum.
  • a nucleic acid sequence also from the genus Taraxacum and optionally a 3 1 UTR from the genus Taraxacum.
  • the same can be applied to the species of the host. Although the plant will carry a transgene, all nucleotide elements thereof are naturally found in the host genus or species (albeit not in this combination), reducing regulatory problems and improving public acceptance.
  • transformants expressing high, constitutive levels of the protein or of the sense and/or antisense transcript (when silencing constructs are used) can be selected by e.g. analysing copy number (Southern blot analysis), mRNA transcript levels (e.g. Northern blot analysis or RT-PCR) or by analysing the presence and level of protein encoded by the nucleic acid sequence (e.g. SDS-PAGE followed by Western blot analysis; ELISA assays, immunocytological assays, etc).
  • the transformants can also be tested for the stability of expression under one or more conditions and those events which retain high, constitutive expression under one or more of the desired conditions can be identified and selected for further use.
  • the transgenic plants can be used in traditional breeding methods, such as crossing, selfing, backcrossing, etc. By selfing the transformants, plants which are homozygous for the transgene can be generated. Breeding procedures are known in the art and are described in standard text books of plant breeding, e.g., Allard, R.W., Principles of Plant Breeding (1960) New York, NY, Wiley, pp 485; Simmonds, N.W., Principles of Crop Improvement (1979), London, UK, Longman, pp 408; Sneep, J. et al., (1979) Tomato Breeding (p. 135-171) in: Breeding of Vegetable Crops, Mark J.
  • Transgenic cells or organisms can also be used in cell cultures (plant cell cultures, bacterial or fungal cell cultures such as yeast cultures, human or mammalian cell cultures, insect cell cultures), for example for the large scale production of recombinant proteins.
  • a cell culture comprising cells comprising a promoter according to the invention.
  • transgenic plant or plant cell comprising the steps of:
  • the regenerated plant (or progeny thereof which retain the transgene) or parts thereof may be used for various purposes, such as in agriculture as such or for molecular farming.
  • the further use depends on the phenotype conferred by the transgene. For example, if the transgenic plant produces high levels of a secondary metabolite, the plants will be grown and the metabolite harvested. Thus, all or part of the plants may be harvested for either human or animal consumption or for industrial purposes, depending on the transgene. Also, different parts of the plant may be harvested for different purposes, e.g. the fruit or seed may be harvested for consumption, while the roots, leaves, stems and/or flowers may be harvested for industrial purposes.
  • the phenotype conferred by the transgene can be tested, e.g. in field trials (e.g. disease or pest resistance tests can be carried out using conventional methods).
  • the plants may be used in conventional agricultural and breeding methods.
  • Figures Figure 1 describes the promoter sequence of the cisprenyltransferase.
  • the 3'ATG is the start codon of cptl
  • Figure 1A demonstrates the Expression of GUS gene from E. coli under control of Pcpti in Taraxacum koksaghyz.
  • Figure 2 describes the promoter sequence of the small rubber particle protein.
  • the 3'ATG is the start codon of srpp3.
  • the method according to the invention can be carried out both with whole plants or parts thereof, wherein the parts of the plants are preferably selected from the group consisting of cells, tissues, organs, roots, stems, branches, leaves, and mixtures thereof.
  • all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.
  • the promoter sequences of cpt (cis-prenyltransferase) and srpp (small rubber particle protein) were amplified by the use of Genome Walker technique (Clontech). Therefore genomic DNA of Taraxacum koksaghyz was isolated by CTAB method (Doyle, 1990) and used for preparing the Genomic Walker libraries according to the manufacturers recommendations. Following primers were designed from parts of the genomic sequences of cpt1 and srpp3, respectively and used in subsequent Genome Walker steps to obtain the corresponding promoter sequences.
  • cpt-promSeq1 5 1 -GAATGCTGTGTGTAAGATCACTTTC-3 ⁇ [SEQ ID 3]
  • cpt-promSeq2 ⁇ '-GTAGCCGCGTAGGCGATCATAAGTGTTTAC-S' [SEQ ID 4]
  • srpp-revGW S'-CTGCAAATTGGCTGAGTAATTGGGCG-S' [SEQ ID 5]
  • the obtained promoter sequences were cloned into the pCRII-TOPO (Invitrogen) and verified by sequencing.
  • the promoter sequences were amplified by PCR using a Pfu-DNA-Polymerase (NEB) with primers including recognition sites for the restricition enzymes Sphl and Xhol (cpt1 prom-Sphl 5'- GCATGCCATTAGTCCCTTCAGTTTGAC-3' [SEQ ID 6]
  • cpt1 prom-Xhol 5'- ATACTCGAGCTTGGTTTAACAAATGAGCC-S' [SEQ ID 7] ; srpp3prom-Sphl S'- TTTGCATGCGACTTGAACTTTTGCCCGAC-S' [SEQ ID 8 , srpp3prom-Xhol ⁇ '- TTTCTCGAGGATCAAGATGTATATGGTTC-S' [SEQ ID 9] ) and ligated via those restriction sites in front of the GUS gene from Escherichia coli into a pUC based plasmid.
  • the binary plasmids were designated as pPcpt1 :GUS and pPsrpp3:GUS and transformed into the Agrobacterium tumefaciens strain EHA105. The integrity of all constructs was verified by sequencing.
  • Taraxacum koksaghyz was transformed by A. tumefaciens-mediated leaf disc infection.
  • A. tumefaciens strain EHA105 carrying a plasmid that contained a promoter reporter gene construct, was cultured in 100 ml induction broth (5 g 1-1 sucrose, 5 g 1-1 peptone, 5 g 1-1 casein hydrolysate, 1 g 1-1 yeast extract, 10 mM MES, 2 mM MgSO4, pH 5.6) containing 50 mg 1-1 kanamycin, 100 mg 1-1 rifampicin.
  • the bacteria were cultured at 28 0 C to stationary phase, then centrifuged, and the pellet was resuspended in coculture medium (4.4 g 1-1 Murashige-Skoog salt solution including vitamins, 10 mM MES, 20 g 1-1 glucose, pH 5.6), supplemented with 200 ⁇ M acetosyringone.
  • Leaf discs ( ⁇ 1 cm2) were punched from the leaves of 6- to 10-week-old T. koksaghyz plants that had been grown under sterile conditions on solid medium (2.2 g 1-1 Murashige-Skoog salt solution, 10 g 1-1 glucose, 8 g 1-1 agar, pH 5.8) and inoculated in the coculture medium with A.
  • tumefaciens for 30 min.
  • the leaf discs were then placed on filter paper for 14-20 h at 26°C.
  • the leaf discs were placed on regeneration medium (4.4 g 1-1 Murashige-Skoog salt solution including vitamins, 18 g 1-1 glucose, 8.5 g 1-1 agar, pH 5.8) supplemented with 2 mg 1-1 6- benzyladenine and 0.1 mg 1-1 naphthalenacetic acid for callus and shoot induction.
  • the elongation of shoots was maintained by the addition of 2 mg 1-1 zeatin, 20 ⁇ g 1-1 naphthalenacetic acid and 20 ⁇ g 1-1 gibberellic acid GA3. Rooting was induced on regeneration medium without hormones, whereas all regeneration media contained 3 mg 1-1 phosphinothricin for selection. All chemicals and reagents were purchased from Duchefa.
  • cis-polyprenyltransferase and small rubber particle protein were originally isolated as partial cDNA fragments (an EST of srpp3 and a homologues EST for another cpt are now available at TIGR). There has not been any entry of the genomic locus of whether cpt1 or srpp3 made to databases, so far. Both genes show homology to the genes from Hevea brasiliensis.

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Abstract

L'invention porte sur des promoteurs pour la cis-phényl transférase ou une protéine des petites particules de caoutchouc, sur des plantes ou cellules de plante ou tissus de plante ou organes transgéniques comprenant un gène chimérique contenant le promoteur, sur des vecteurs contenant le promoteur et sur l'utilisation du promoteur dans l'expression d'une séquence d'acide nucléique dans une plante, en particulier des plantes lactifères.
PCT/EP2008/010563 2008-01-31 2008-12-10 Promoteurs de la biosynthèse du caoutchouc à partir de taraxacum koksaghyz et leur utilisation WO2009095059A1 (fr)

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CN104542303A (zh) * 2015-01-28 2015-04-29 云南农业大学 一组通关藤快速繁殖培养基
EP3059315A1 (fr) * 2015-02-23 2016-08-24 Sumitomo Rubber Industries, Ltd. Vecteur comprenant un promoteur spécifique et gène codant pour une protéine spécifique, plante transgénique dans laquelle le vecteur a été introduit et procédé permettant d'améliorer la production de polyisoprénoïde par introduction du vecteur dans une plante
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EP3059317A1 (fr) * 2015-02-23 2016-08-24 Sumitomo Rubber Industries, Ltd. Vecteur comprenant un promoteur spécifique et gène codant pour une protéine spécifique, plante transgénique dans laquelles le vecteur a été introduit et procédé permettant d'améliorer la production de polyisoprénoïde par introduction du vecteur dans une plante
EP3059316A1 (fr) * 2015-02-23 2016-08-24 Sumitomo Rubber Industries, Ltd. Vecteur comprenant un promoteur spécifique et protéine spécifique codant un gène, plante transgénique dans laquelle le vecteur a été introduit, et procédé permettant d'améliorer la production de polyisoprénoïde par introduction du vecteur dans une plante
WO2016204503A1 (fr) * 2015-06-15 2016-12-22 아주대학교 산학협력단 Procédé pour la production de caoutchouc naturel au moyen de micro-organisme recombinant
US9890262B2 (en) 2012-03-06 2018-02-13 Bridgestone Corporation Processes for the removal of rubber from non-hevea plants
US10023660B2 (en) 2012-05-16 2018-07-17 Bridgestone Corporation Compositions containing purified non-hevea rubber and related purification methods
US10113011B2 (en) 2008-04-14 2018-10-30 Bridgestone Corporation Process for recovering rubber from natural rubber latex
US10132563B2 (en) 2012-06-18 2018-11-20 Bridgestone Corporation Methods for the desolventization of bagasse
US10138304B2 (en) 2012-06-18 2018-11-27 Bridgestone Corporation Methods for increasing the extractable rubber content of non-Hevea plant matter
US10287367B2 (en) 2013-09-11 2019-05-14 Bridgestone Corporation Process for the removal of rubber from TKS plant matter
US10471473B2 (en) 2012-06-18 2019-11-12 Bridgestone Corporation Systems and methods for the management of waste associated with processing guayule shrubs to extract rubber
US10775105B2 (en) 2018-11-19 2020-09-15 Bridgestone Corporation Methods for the desolventization of bagasse

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CN104542303B (zh) * 2015-01-28 2016-05-18 云南农业大学 一组通关藤快速繁殖培养基
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EP3059316A1 (fr) * 2015-02-23 2016-08-24 Sumitomo Rubber Industries, Ltd. Vecteur comprenant un promoteur spécifique et protéine spécifique codant un gène, plante transgénique dans laquelle le vecteur a été introduit, et procédé permettant d'améliorer la production de polyisoprénoïde par introduction du vecteur dans une plante
EP3059317A1 (fr) * 2015-02-23 2016-08-24 Sumitomo Rubber Industries, Ltd. Vecteur comprenant un promoteur spécifique et gène codant pour une protéine spécifique, plante transgénique dans laquelles le vecteur a été introduit et procédé permettant d'améliorer la production de polyisoprénoïde par introduction du vecteur dans une plante
EP3059318A1 (fr) * 2015-02-23 2016-08-24 Sumitomo Rubber Industries, Ltd. Vecteur comprenant un promoteur spécifique et gène codant pour une protéine spécifique, plante transgénique dans laquelles le vecteur a été introduit et procédé permettant d'améliorer la production de polyisoprénoïde par introduction du vecteur dans une plante
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KR20160148470A (ko) * 2015-06-15 2016-12-26 아주대학교산학협력단 재조합 미생물을 이용한 천연고무의 생산 방법
WO2016204503A1 (fr) * 2015-06-15 2016-12-22 아주대학교 산학협력단 Procédé pour la production de caoutchouc naturel au moyen de micro-organisme recombinant
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