WO2010010207A1 - Promoteur constitutif de solanum lycopersicum - Google Patents

Promoteur constitutif de solanum lycopersicum Download PDF

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WO2010010207A1
WO2010010207A1 PCT/ES2009/000375 ES2009000375W WO2010010207A1 WO 2010010207 A1 WO2010010207 A1 WO 2010010207A1 ES 2009000375 W ES2009000375 W ES 2009000375W WO 2010010207 A1 WO2010010207 A1 WO 2010010207A1
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plant
promoter
fruit
expression
interest
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Spanish (es)
Inventor
Diego Orzaez Calatayud
Leandro Hueso Estornell
Benito Pineda Chaza
Teresa ANTÓN MARTÍNEZ
Vicente Moreno Ferrero
Antonio Granell Richart
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Fundacion Para El Desarrollo de la Investigacion En Genomica Y Proteomica
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Fundacion Para El Desarrollo de la Investigacion En Genomica Y Proteomica
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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/08—Fruits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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)

Definitions

  • the present invention relates to a CNH1 promoter sequence (SEQ. ID.
  • the invention also relates to nucleic acid constructs, vectors, plant cells and transgenic plants comprising the promoter thus as to the seeds thereof.
  • the promoter is useful when fused to appropriate DNA sequences to direct the sense gene expression, miRNA, antisense or RNAi in all the organs of the plant and especially with high levels in the fruit.
  • Promoters are regulatory elements that direct the expression of genes. Both constitutive promoters and regulators are used to direct gene expression in transgenic organisms including plants.
  • the constitutive promoters direct the expression in the majority or in all tissues, and are useful when high levels of production of a gene product are desired throughout the plant.
  • the 35S promoter of the cauliflower mosaic virus (VMC) is the promoter that is most frequently used to direct high levels of constitutive expression in plants.
  • Regulatory promoters such as inducible and tissue specific promoters, are used to direct spatial or temporally specific expression or expression in response to environmental factors.
  • CaMV35S promoter is the most frequently used promoter in plant biotechnology (US Food and Drug Administration for Food Safety and Nutrition, http://www.cfsan.fda.gov/ ⁇ lrd/biocon. html).
  • Different versions derived from this promoter have been used (Fang, RX, Nagy, F., Sivasubramaniam, S. and Chua, NH 1989.
  • This promoter has been shown to be useful for directing good expression levels when cloned against a gene of interest, in almost all dicotyledonous plants in which it has been tested (including Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Zea mays and Oryza sativa, etc.) (US Food and Drug Administration for Food Safety and Nutrition, (http://www.cfsan.fda.gov/ ⁇ lrd/biocon.html).
  • CmYLCV Certrum yellow leaf curling viru s
  • the genetically modified plant with cisgenes is a cisgenic plant ⁇ Cisgenic plants are similar to traditionnally bred plants. Schouten, HJ. , Krens, FA, Jacobsen, E., 2006. EMBO Reports 7: 750-753; "Cisgenic” as a product designation, Schubert D & Williams D. 2006. Nature Biotechnology 24: 1327-1329. Unlike a transgene, which is defined as a gene or DNA sequence of non-sexually compatible species or a DNA sequence or synthetic gene. The corresponding genetically modified plant that contains transgenes is a transgenic plant.
  • the promoter described herein can be used in all plant species as an alternative to CaMV35S but it is of special application in the Solanum genus to confer constitutive expression of genes or DNA sequences of interest with particularly high levels in the fruit, in All stages of development.
  • cisgenesis or intragenesis any desired characteristic due to a dominant allele available in the gene pool in sexually compatible species can be isolated, subsequently subjected to the control of the CNH promoter and introduced into tomato or related species to confer the characteristic of interest.
  • the present invention provides a promoter that directs high levels of gene expression or DNA sequences in plants, especially high in the fruit.
  • This promoter derives from a gene of unknown function of Solanum lycopersicum that is expressed ubiquitously throughout the plant.
  • This tomato promoter can be from special interest to direct the expression of genes or DNA sequences of interest of related species within Solanaceae I Solanum (the tomato belongs to this genus) so that high levels of expression (similar to CaMV35S in the fruit) can be achieved without the need for use heterologous promoters and therefore it is of interest for the directed introduction of genes "within Solanaceae / Solanum using a strategy in which all sequences are from Solanaceae / Solanum".
  • the promoter of the invention can direct high levels of constitutive expression of DNA sequences or heterologous genes in plants, especially when higher levels are desired in the fruit.
  • the promoter of the present invention is useful when expression levels similar to those obtained by the CaMV 35S promoter are desired in the fruit during different stages of its development, but not so high in the rest of the plant.
  • the present invention relates to an isolated promoter derived from a Solanum lycopersicum tomato gene with unknown function.
  • this gene is the CNH gene.
  • the promoter has at least 70% identity with the sequence of SEQ. ID. NO: 2.
  • This promoter is of special utility fused to appropriate DNA sequences, to direct the constitutive sense gene expression, miRNA, antisense or RNAi in most of the tissues of main plants during all their life cycles, but with especially high levels in the fruit .
  • This tomato promoter may be of special interest to direct the expression of genes or DNA sequences of interest of related species within the Solanaceae / Solanum species to which the tomato belongs, being able to obtain high levels of expression (similar to those obtained in the fruit through the CaMV35S promoter, but smaller than those obtained through it in the rest of the tissues of the plant), without the need to use heterologous promoters. That is, the promoter of the invention has interest in the production of "targeted introgression of genes" in Solanaceae / Solanum using "the all Solanaceae / Solanum strategy".
  • the promoter of the invention derived from CN1H allows the expression in plants, preferably in the fruit, not only of endogenous genes or DNA sequences but also of heterologous genes or DNA sequences, in orientation sense or antisense, to achieve overexpression or silencing of the expression of said genes.
  • These genes can be plant-specific genes or DNA sequences of the fruit, or hetorologous genes or sequences whose expression is desired in the fruit.
  • the CNH 1 promoter of the invention is effective in the Tomato lycopersicum plant from which it is derived, in other species of the Solanaceae I Solanum genus, as well as in other plants that bear fruits.
  • the promoter of the invention is ideal for applications in which a ubiquitous expression of a gene or a DNA sequence (endogenous or heterologous) is desired in the plant, but with higher levels in the fruit, when similar expression levels those obtained by the CaMV35S promoter are desired in the fruit but not so high in the rest of the plant, and when more than one constitutive promoter is necessary to express two or more recombinant proteins in a plant in a concerted manner and preferably in the fruit
  • the promoter of the invention is useful in strategies of the "biofactories” type in which the fruit is used as a platform for the recombinant production of proteins that themselves harbor biological or therapeutic activity when administered orally, or that regulate or catalyze the production of other metabolic compounds with biological or therapeutic activity by mouth (oral vaccines, tolerogens, biocides, neutralizing antibodies, adjuvants, antioxidant compounds, vitamins, aromas, dietary supplements); in the production of multiproteic complexes that require to express in concerted form two or more genes with different constitutive promoters to avoid gene rearrangements and / or gene silencing processes; in the acquisition of pathogen resistance phenotypes, for the modification of the architecture of the fruit giving the plant and the fruit different properties depending on the gene or nucleotide sequence that is expressed under its control.
  • Promoter is a regulatory element that directs the expression of genes.
  • Constitutive promoter refers to those promoters that direct expression in most or all of the tissues and are useful when high levels of production of a gene product are desired throughout the plant.
  • Regulated promoters, tissue-specific and inducible promoters are those used to direct the specific spatial or temporal expression, or the expression in response to environmental factors.
  • Genes and DNA sequences of interest refers to those genes and sequences that code for peptides of interest that can be used together with the CNH1 promoter of the invention. They refer to sequences and genes of Solanum lycopersicum plants, of plants of the Solanum genus or any heterologous, or synthetic gene or sequence.
  • Said genes and sequences include, but are not limited to genes involved in the modification of maturation; genes involved in conferring fungal resistance; genes related to plant pathogenesis; genes involved in conferring resistance against insects; genes that are effective in the modulation of flowering, aroma, color modification, enzymes or other catalytic products (such as: ribozymes or catalytic antibodies that modify cellular processes in plants), ethylene production (such as: antisense molecules, enzymes that they degrade precursors of ethylene biosynthesis, catalytic products or silencing molecules), genes for the control of fungi, production or levels of plant hormones, the cell cycle or cell division, and accumulation of sucrose (such as the gene of Ia sucrose phosphate synthase).
  • Genes and sequences of interest also refers to those genes and sequences that code for peptides or proteins that can be used in "molecular pharming" for the recombinant production of proteins that themselves harbor biological or therapeutic activity or that regulate or catalyze the production of other metabolic compounds with biological or therapeutic activity (oral vaccines, tolerogens, biocides, neutralizing antibodies, adjuvants, antioxidant compounds, vitamins, aromas, dietary supplements).
  • Heterologous Gene or Sequence Gene or nucleotide sequence of synthetic origin or obtained from the genome of animals or plants of a genus other than Solanum.
  • Endogenous Gene or Sequence Gene or nucleotide sequence originally contained in whole or in part in the genome of one or more species of the Genus Solanum
  • Genetically transformed plant refers to those plants of Solanum lycopersicum, of the genus Solanum, or any fruit plant that has been transformed with a gene construction that comprises the promoter of the invention fused to the gene or DNA sequence of interest.
  • Molecular pharming refers to the modification, through genetic engineering techniques, of the substantial content of the plants in order to enrich it in one or more compounds that give them added value.
  • vaccine subunits of viral or bacterial origin are included, peptides or proteins with tolerogenic activity, peptides, proteins with adjuvant or immunomodulatory activity, biocides, immunoglobulins, enzymes or regulatory proteins that catalyze or potentiate the production of metabolites with biological or therapeutic activity, etc.
  • the object of the present invention refers to the promoter of the CNH1 gene, which comprises the sequence SEQ. ID. NO: 2 or at least one sequence that has 70% identity with SEQ. ID. NO: 2, which directs the expression of sequences or genes of interest at high levels in all tissues of the plant, especially in the fruit, during the different stages of development.
  • the expression directed by this promoter in the fruit is similar to that provided by the constitutive promoter CaMV35S, and at least an order of magnitude lower, preferably 3 to 6 times smaller, in the rest of the plant.
  • Another object of the invention relates to nucieotidic constructs that comprise the promoter of the invention operatively fused to a gene or DNA sequence that encodes a peptide of interest.
  • said peptide of interest has application in "molecular pharming". In another particular embodiment, the peptide of interest has application in the production of a medicament.
  • said DNA gene or sequence encodes endogenous proteins or peptides of the Solanum species. In another particular embodiment, said DNA gene or sequence codes for proteins or peptides heterologous to the Solanum species (vegetable, animal or synthetic proteins). In another particular embodiment, said DNA gene or sequence codes for immunoglobulins, specifically for lnmunoglobulin A.
  • Another object of the invention relates to the vectors containing the nucleotide sequence of the invention.
  • Another object of the invention is directed to the cells of plants that contain the nucleotide construction of the invention. Another object of the invention relates to genetically modified plants that comprise the construction of the invention.
  • said plants are Solanum lycopersicum plants, or plants of the Solanum genus.
  • said plants comprise any fruit plant.
  • Another object of the invention relates to the seeds of genetically modified plants.
  • Another object of the invention relates to the fruit of genetically modified plants.
  • Another object of the invention relates to the use of the promoter to direct the expression of genes of interest in plants characterized in that said expression occurs at high levels throughout the plant, with higher levels in the fruit during the different stages of development.
  • Another object of the invention relates to the use of the promoter to direct the expression of genes or DNA sequences of interest in plants when more than one constitutive promoter is necessary to express two or more recombinant proteins in concert and preferably in the fruit.
  • Another object of the invention relates to the use of the promoter in "bio-factories" type strategies.
  • Another object of the invention relates to SEQ. ID. NO: 2.
  • the invention provides the methods for obtaining the transgenic plants and the progeny thereof comprising the construction and parts of such plants,
  • Figure 1 Shows the relative expression values of the CNH1 mRNA in different tissues of the tomato plant, calculated by quantitative PCR. In this representation, the mRNA levels observed in the ovaries in the stage of the anteis were arbitrarily assigned a value equal to one.
  • Figure 2. Represents the expression vector pCNH1 :: GFPGUS. The expression vector includes the CNH1 promoter in the 5 'sense of the GFP-fused sequences.
  • GUS and kanamycin resistance for seed selection between the right and left edges (RB and LB respectively).
  • the resistance to spectinomycin is for the selection of cultures.
  • Figure 3 Shows the values resulting from the quantification of the specific glucuronidase (GUS) activity conferred by the construction pCNH1: GFPGUS in different organs of the tomato plant. These values are compared with the specific activity conferred by the 35S :: GFPGUS construction in another plant of the same species.
  • the specific activity of glucuronidase (GUS) present in the crude extracts of plant tissue is represented as nanomoles of the methyl umbeliferone product generated by the GUS enzyme per minute and per milligram of total protein present in the extract.
  • Figure 4 Shows a histochemical staining of GUS activity in tomato sections at different stages of development. From left to right, fruits in expansion (15 days before) are shown, fruits in the green-mature stage (35 days after before), fruits in the stadium and red fruits respectively.
  • Section A shows fruits transformed with the construction pCNH1 :: GFPGUS.
  • Section B shows fruits transformed with the CaMV35S :: GFPGUS construction.
  • Figure 5 Shows in relative values and through experiments of transient expression in fruit, the potency of the CNH 1 promoter to direct the accumulation of the green fluorescent protein (GFP) in green and ripe fruit and compares it with the activity of the 35S promoter in same organs and physiological stages.
  • the fruits were agroinjected with either a mixture (1: 1) of the CaMV35S: GFP / GUS and CaMV35S: DsRed (samples labeled 35S) or with a mixture (1: 1) of the pCNH1: GFPGUS and CaMV35S constructions: DsRed (samples labeled CNH1).
  • FIG. 5A Scan image of samples of 35S and NH fruits that show green fluorescence as a result of a transient transformation.
  • Figure 5B Green / red fluorescence ratio of different samples as a measure of the relative strength of the promoter in each physiological stage.
  • MG fruits collected in the green-mature stage;
  • Br fruits collected in the stadium pintón.
  • Figure 6 Represents a nucleotide construction in which the CNH1 promoter is used for recombinant tomato production, preferably in fruits and in stages prior to maturation, of an immunoglobulin A with binding activity against the VP8 * peptide of rotavirus.
  • the immunoglobulin A consists of two light and heavy polypeptide chains. The heavy chain is placed under the control of a constitutive promoter derived from CaMV35S, while the CNH 1 promoter directs the expression of the light chain.
  • the construction consists of a vector derived from pK7GW with resistance to Spectinomycin (Sm / SpR) and which includes the following elements: RB: right edge; LB: left edge; KanR: Kanamycin resistance gene as a selection marker in the transformation process; attB1, attB4, attB3 and attB2: small recombination sequences by-product of the cloning mechanism used in the construction of the fragment (homologous recombination mediated by clone LR); 35S: double constitutive promoter derived from CaMV35S; SP: signal peptide to direct the entry of the subsequent protein in the cell endomembrane system, where immunoglobulins are more stable; IgH: nucleotide sequence of a heavy chain of a human immunoglobulin with affinity for VP8 *, NosT: terminator of the transcription of the nopaline synthase gene; CNH1: the promoter of the present invention; IgL: the nucleo
  • FIG. 7 Western blotting under denaturing, non-reducing conditions of raw extracts of tomato fruits agroinfected with the T-DNA fragment of Figure 6 (lane 1); agroinfected fruits with an unrelated construction (lane 2); leaves agri-infected with the T-DNA fragment described (lane 3)
  • White bars CNH1 containing T-DNA construction.
  • Black bars construction of unrelated T-DNA.
  • FIG. 9 Sequence of the CNH1 promoter fragment used for the construction of pCNH1-GUS / GFP (SEQ. ID. NO: 2). The figure shows how the sequence of the GSP3 primer used for the amplification without the recombination fragment is highlighted.
  • a tomato fruit cDNA library was constructed using the lambda ZAPII cDNA library construction kit (Stratagene, LaJoIIa, Calif.). The cDNA library was converted into a plasmid library by mass cleavage, and bacterial clones were plated and replicas of this library corresponding to about 5,000 clones were obtained on filters (following Maniatis, Sambrook and Fritsch 1982).
  • Each replica filter was then hybridized with cDNA probes prepared from two samples representing fruits gathered at different stages of development and collected vegetative tissues of Solanum lycopersicum.
  • the PCR random sequence primer technique (RP-PCR) described by Li Z and Thomas TL was used.
  • PEH an Embryo-Specific Zinc Finger Protein Gene Required for Heart-Stage Embryo Formation in Arabidopsis, 1998, Plant CeII 10: 383
  • Hybridization of each colony to each probe was used to determine the colonies that represent putative tissue constituent or specific genes. Putative constitutive clones were identified and subjected to further characterization. The identity of each clone was determined by DNA sequencing and database searches.
  • CNH 1 The clone called CNH 1 was selected from the putative constitutive clones chosen for further characterization based on their high levels of expression, revealed by RNA dot blot analysis. Northern blot analysis showed that CNH1 is expressed at high levels in the set of both vegetative and fruit samples. Quantitative RT-PCR confirmed that CNH1 was expressed in all tissues analyzed, but especially in the fruit ( Figure 1). Figure 1 shows the relative expression levels of CNH 1 in different tissues, taking as reference the expression levels in ovaries in the antecedents.
  • the first stage was to construct sets of genomic DNA fragments linked to adapter, not cloned, called "Genome Walker libraries".
  • the starting DNA must be very clean and have a high average molecular weight, since it was considered the most suitable method to obtain this DNA from young tissues such as those present in tomato seedlings.
  • each batch of digested genomic DNA was ligated separately to a designed adapter.
  • the adapters were provided by the kit.
  • the first or primary PCR required an external adapter primer (AP1, SEQ. ID. NO: 4) that was provided in the kit, and an external gene specific primer (GSP1, SEQ. ID. NO: 5) designed from of the known 5'UTR gene sequence.
  • the primary PCR mixture was then diluted and used as a template for a secondary or "nested” PCR with the nested adapter primer (AP2, SEQ. ID. NO: 6) also provided in the kit, and a gene specific primer " nested "(GSP2, SEQ. ID. NO: 7), which was designed by taking a part of the GSP1 sequence (SEQ. ID.
  • Gateway cloning systems based on homologous recombination, are have become a popular alternative to traditional cloning methods based on T4 ligase for the construction of plant expression cassettes ("GATEWAY vectors for Agrobacter ⁇ um-mediated plant transformation", Karimi M 1 Inzé D and Depicker A., Trends Plant Sci, 2002, 7: 192-195).
  • GATEWAY vectors for Agrobacter ⁇ um-mediated plant transformation Karimi M 1 Inzé D and Depicker A., Trends Plant Sci, 2002, 7: 192-195.
  • a PCR reaction was performed using two highly purified and long primers (attB1-GSP3 (SEQ. ID. NO: 8, 10) and attB2-GSP2 (SEQ; ID. NO: 9, 7)) each containing of them a specific recombination sequence.
  • BP reaction a vector was formed containing the promoter sequence flanked by the attl_1 and attl_2 recombination sites, and called “pEntry vector”.
  • LR reaction a second recombination reaction
  • the fluorometric GUS test confirmed the expression pattern of this promoter, demonstrating the expression of GUS, in all plant tissues, but especially in fruit tissues.
  • the transient gene expression mediated by Agrobacter ⁇ um was chosen through agri-injection in tomato fruits (Orzaez D, Mirabel S, Wieland WH, Granell A. (2006), Agroinjection of tomato fruits.A tool for rapid funtional analysis of transgenes directly in fruit, Plant Physiol. 2006 Jan; 140 (1): 3-1 l).
  • pCNH1 :: GFPGUS was introduced into A. tumefaciens C58 for these transient expression assays and injected together with solution of pBIN 35S: DsRed transformed by Agrobacter ⁇ um. Pieces of different fruits were scanned to obtain relative GFP / DsRed fluorescence values.
  • a double stranded DNA containing the promoter of the invention can be introduced into the genome of the plant by any suitable methodology. This includes the infection of plant tissue with cultures of
  • Agrobacter ⁇ um transformed with plasmids derived from Agrobacter ⁇ um tumefaciens Agrobacter ⁇ um transformed with plasmids derived from Agrobacter ⁇ um tumefaciens.
  • liposomes include liposomes, electroporation, chemical treatments, particle bombardment or other alternative methods to introduce DNA into the cells, or the transformation mediated by viruses or pollen.
  • An especially useful vector is pKGWO (Karamini e ⁇ al, 2002), derived from pPZP200 (Hajdukiewicz e ⁇ al, 1994), which includes a Kanamycin resistance marker (Hellens e ⁇ al, 2000) and a cassette gateway (Invitrogen life technologies) for facilitate cloning by homologous recombination, all between the sequences known as right border and left border and that delimit the integration fragment in the genome of the plant.
  • Other vectors known in the state of the art could be used.
  • the cells are regenerated in whole plants using a regeneration protocol appropriate to the host plant.
  • the promoter of the invention is useful in applications in which the strong constitutive expression of a gene or a DNA sequence (endogenous or heterologous) is desired in all tissues of the plant, especially in the fruit at different stages of development, when expression levels similar to those obtained by the 35 S promoter are desired in the fruit but not so high in the rest of the plant.
  • said promoter may be useful in "molecular pharming approaches "for oral immunization, as a factory of proteins that harbor biological or therapeutic activity by themselves or that regulate or catalyze the production of other metabolic compounds with biological or therapeutic activity by mouth (oral vaccines, tolerogens, biocides, antibodies neutralizers, adjuvants, antioxidant compounds, vitamins, aromas, dietary supplements); in the production of multiproteic complexes that require to express in concert two or more genes with different constitutive promoters to avoid gene rearrangements and / or gene silencing processes; in the acquisition of phenotypes of resistance to pathogens, for the modification of the architecture of the fruit giving the plant and the fruit different properties depending on the gene or nucleotide sequence that is expressed under its control.
  • genomic DNA was excreted from tomato seedlings grown in the dark.
  • the extraction of genomic DNA was carried out using the Dneasy Plant (Qiagen) minikit and following the manufacturer's protocol.
  • the DNA fragments of each digestion group were ligated to 48 bp adapters (SEQ. ID. NO: 3) on both sides. They were carried out ligations T4 DNA ligase using a reaction during night Ia 16 0 C and 7O 0 C reactions were stopped for 5 minutes.
  • AP1 SEQ. ID. NO: 4
  • AP2 SEQ. ID. NO: 6
  • the first PCR reaction required the AP1 primer and a first gene specific primer (GSP1) (SEQ. ID. NO: 5) that was designed so that its sequence started at position -4 and contained the ATG codon of CNH1.
  • GSP1 first gene specific primer
  • the PCR product was diluted 1: 50 and used for a second PCR reaction using the nested adapter primer (AP2) (SEQ. ID. NO: 6) and a second gene specific primer (GSP2) designed from Ia position -37 (SEQ. ID. NO: 7).
  • AP2 nested adapter primer
  • GSP2 second gene specific primer
  • the Advantage Genomic Polymerase (Clontech) mixture was used for PCR reactions.
  • GSP3 oligoceador was designed in the 3 'direction of the adapter sequence for add the attB1 recombination sequence (SEQ. ID. NO: 8) to the authentic sequence in the 5 'direction.
  • I KNOW THAT. ID. NO: 2 shows the sequence resulting from 3657 bp of the promoter fragment used in the construction of pCNH1-GUS / GFP to direct the expression of endogenous or heterologous genes or DNA sequences in plants as follows.
  • the 3657 bp promoter fragment was amplified by PCR using a portion of the GSP2 primer bound to an attB2 recombination sequence (SEQ. ID. NO: 9) and GSP3 (SEQ. ID. NO: 10).
  • the GSP3 primer sequence used for amplification without the recombination fragment is highlighted in Figure 9.
  • the fragment thus obtained was recombined in a BP reaction with the vector pDONR221 to provide a pEntry vector.
  • a reaction at 25 0 C was performed according to the manufacturer 's instructions (Invitrogen), using between 15 and 150 ng of the PCR product, 1 ul of pDONR221, 1X TE buffer and BP Clonasell reaction mixture.
  • reaction 1X LR reaction was performed according to the manufacturer at 25 0 C, using 200 ng of vector and 300 ng pENTRY vector PDestination TE buffer and LR
  • Clonasell The binary vector pKGWFS7.0 (Karimi et al, 2002) was used as the pDestination vector, which was considered more suitable for a promoter activity assay due to the dual GFP-GUS (SEQ. ID. NO: 11) which was fused into the 3 'direction of the attR2 recombination site.
  • the selection of the transformed clones was carried out in an agar medium plus selective LB spetinomycin.
  • GFP-GUS is shown in Figure 2.
  • Figure 3 shows the levels of GUS enzymatic activity obtained in different organs of a plant transformed with the T-DNA CNH1 :: GUS / GFP and where therefore the GUS gene is regulated by the promoter of the invention
  • CNH1 confers expression to GUS in all the organs of the plant, however this expression is much stronger in the fruit throughout its development, being in this organ its very similar expression levels to which the reference promoter CaMV 35S confers.
  • the maximum levels of GUS activity conferred by CNH1 are 24 nanomoles of methylumbelliferone produced per minute and milligram of total protein in raw extracts of tomato fruit in green-mature stage.
  • the CaMV 35S and CNH1 promoters differ in their ability to discriminate between the fruit and the other tissues of the plant. Therefore, it is concluded that the CNH 1 promoter is useful for achieving gene expression ubiquitously throughout the plant but especially high (similar to CaMV35S) in the fruit.
  • the CNH1 :: GUS / GFP cassette (SEQ. ID. NO: 13) introduced into tomato plants as described in example 1, is detected by histochemical staining in the fruit following the protocol described by Jefferson ef al., id., in order to determine the distribution of the GUS activity conferred by CNH1 in the different tissues of the fruit. From left to right, the figure shows an expanding fruit (15 days before), a fruit in a green-mature stage (35 days before), a fruit in the stage and a red fruit respectively.
  • Figure 4a shows that the distribution of GUS staining directed by CNH 1 extends to all tissues and all stages of development studied.
  • FIG. 4b shows, in a similar image, the GUS activity conferred by the constitutive promoter CaMV35S in the fruit included in the 35S :: GUS / GFP construction (SEQ. ID. NO: 14).
  • the comparison of both images proves that CNH1 can be used analogously to CaMV35S as a constitutive promoter in tomato fruit. From what is stated in Figure 4, it is concluded that the CNH 1 promoter is ideal as an endogenous promoter for the expression of recombinant proteins in the fruit when a ubiquitous and constitutive expression in this organ is intended, since its expression includes all tissues and the different stages of development, analogously to what is observed with the use of a constitutive reference promoter such as CaMV35S.
  • the use of the CNH1 promoter is therefore very convenient when a promoter is required that facilitates continued accumulation and high levels of a gene product in the fruit, especially if this promoter is to be an endogenous promoter.
  • the CNH 1 promoter is useful for directing the expression of recombinant proteins, and in particular of the green fluorescent protein (GFP), in transient fruit expression assays.
  • the transient expression assays described by Orzáez et al (2006), are useful for determining the viability of a particular gene construct for expression in fruit.
  • Transient expression assays have limited transformation efficiency and therefore require constructions with promoters that confer strong expression on the different tissues of the fruit, since otherwise the product of the recombinant gene cannot be detected.
  • a test of the suitability of a promoter for use in transient expression tests in fruit consists in determining its ability to direct detectable and quantifiable expression levels of the green fluorescent protein.
  • the transient expression of GFP conferred by CNH1 was detected in a fluorescence scanner (figure 5 left), and compared with that conferred by CaMV35 in the CaMV35-GUS / GFP construction (figure 5th right).
  • the CaMV35: DsRed construction was used as internal standard, which directs the constitutive expression of a red fluorescent protein. This construction was used as an internal standard to normalize the variations in green fluorescence due to the efficiency of the transient transformation.
  • the fluorescence emitted by the agro-injected fruits was measured at four days post injection (green-ripe) or eight days post injection (representing stages of pinón).
  • This example shows the joint use of the 35S promoters and the promoter of the invention CNH1 for the joint expression in the same T-DNA of a human immunoglobulin A (IgA) against rotavirus.
  • IgA performs a passive immunoprotection function in the mucous membranes.
  • Edible fruits can be an excellent platform for the production of recombinant IgAs against enteropathogenic viruses, reducing both production and purification costs.
  • the IgAs are formed by two polypeptide chains, light and heavy, and therefore their production in fruit requires the concerted expression of both genes in the fruit at high levels.
  • the T-DNA of the present example is constructed by sequentially incorporating the constructions of the heavy and light chain into the expression vector pK7GW (Karimi et al 2002).
  • the nucleotide sequences of the constant region of the heavy chain of the human immunoglobulin A isotype alpha 1 (SEQ. ID. NO: 15) (IgHalphai) and of the constant region of the human light chain isotype lambda (Iglambdal) SEQ ID NO: 16
  • SEQ. ID. NO: 15 the constant region of the human light chain isotype lambda
  • Iglambdal Iglambdal
  • SEQ ID NO: 16 are introduced by homologous recombination into the vector pDNOR221 (Invitrogen), generating the vectors pENTRIgHalpha and pENTRIgLambda respectively.
  • the resulting vector contains a T-DNA that is shown in Figure 6 (SEQ. ID. NO: 19) and that incorporates the necessary elements for the expression in fruit of a human IgA against rotavirus:
  • a fragment of T- was constructed Agrobacterium tumefaciens DNA containing the following elements from 5 'to 3': (1) the integrative left border (SEQ. ID. NO: 20); (2) a complete cassette for kanamycin resistance in plants (SEQ.ID.NO: 21); (3) an attB1 recombination site as an LR recombination byproduct (SEQ. ID. NO: 22); (4) a double 35S promoter (SEQ. ID.
  • T-DNA described above was transiently transferred to tomato fruit cells using the agroinjection procedure (Orzaéz et al 2006).
  • the presence of recombinant human IgA against rotavirus was detected in raw extracts of tomato fruits in Western blot and ELISA.
  • Figure 8a shows the detection in fruit (line 1) by Western blotting of the heavy and light IgA chains. IgA was not detected in control fruits agroinfiltrated only with CamV35S: GFP / GUS, while the expression of both chains was undetectable in tomato leaf agroinfiltrated with the same T-DNA.
  • Figure 8b shows how raw extracts of agro-infiltrated tomatoes with the T-DNA of Figure 7 are capable of specifically binding the rotavirus antigen against which they were initially selected (VP8 *). Undiluted extracts and 1:10 dilutions of the extracts reacted strongly against the antigen in ELISA assays (white bars), while control fruit extracts agroinjected with the 35S construction: GFP / GUS show only a basal signal on plates upholstered with the antigen . It is therefore demonstrated that the CNH 1 contest allows the expression of functional protein complexes in the fruit.

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Abstract

La présente invention concerne une séquence de promoteur (SEQ. ID. N0:2) dérivée d'un gène de Solanum lycopersicum dont la fonction est inconnue et qui est désigné par CNH1. L'invention concerne des constructions nucléotidiques, des vecteurs, des cellules de végétaux et des plantes transgéniques qui comprennent le promoteur selon l'invention, ainsi que des graines de celles-ci. Le promoteur constitutif selon l'invention est utilisé pour l'expression génique sens, micro-ARN, antisens ou ARNi des séquences d'ADN avec lesquelles il fusionne, dans tous les organes de la plante, avec des niveaux particulièrement élevés dans le fruit. Le promoteur présente un intérêt particulier lorsque des niveaux d'expression similaires à ceux obtenus pour le promoteur CaMV 35S sont souhaités dans le fruit au cours de divers stades de son développement, mais pas de façon si élevée dans le reste de la plante, notamment dans Solanaceae/Solanum, ce qui permet d'éviter l'utilisation de promoteurs hétérologues.
PCT/ES2009/000375 2008-07-24 2009-07-17 Promoteur constitutif de solanum lycopersicum Ceased WO2010010207A1 (fr)

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ES200802207A ES2332690B1 (es) 2008-07-24 2008-07-24 Promotor constitutivo de solanum lycopersicum.

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997046690A1 (fr) * 1996-06-07 1997-12-11 Zeneca Limited Renforcement de l'expression genique
US6127179A (en) * 1996-04-17 2000-10-03 Dellapenna; Dean Gene promoter for tomato fruit
US6340748B1 (en) * 1999-09-28 2002-01-22 Seungil Ro Promoter of the tomato expansin gene LeExp-1

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6127179A (en) * 1996-04-17 2000-10-03 Dellapenna; Dean Gene promoter for tomato fruit
WO1997046690A1 (fr) * 1996-06-07 1997-12-11 Zeneca Limited Renforcement de l'expression genique
US6340748B1 (en) * 1999-09-28 2002-01-22 Seungil Ro Promoter of the tomato expansin gene LeExp-1

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