WO2004101614A1 - Sequences de type promoteur provenant des genes d'heveine de l'espece hevea brasiliensis - Google Patents

Sequences de type promoteur provenant des genes d'heveine de l'espece hevea brasiliensis Download PDF

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WO2004101614A1
WO2004101614A1 PCT/IB2004/001904 IB2004001904W WO2004101614A1 WO 2004101614 A1 WO2004101614 A1 WO 2004101614A1 IB 2004001904 W IB2004001904 W IB 2004001904W WO 2004101614 A1 WO2004101614 A1 WO 2004101614A1
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polynucleotide
hevein
promoter
sequence
plant
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PCT/IB2004/001904
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Arokiaraj Pappusamy
Valérie PUJADE-RENAUD
Heddwyn Jones
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Centre De Cooperation Internationale En Recherche Agronomique Pour Le Developpement (Cirad)
Malaysian Rubber Board (Mrb)
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Publication of WO2004101614A1 publication Critical patent/WO2004101614A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems

Definitions

  • the present invention relates to isolation of DNA sequences from the Hevea brasiliensis containing the promoter and regulatory region of hevein genes and to demonstrate their functionality in Hevea as well as in heterologous systems.
  • the present invention also provides a recombinant expression cassette comprising the hevein promoter and a heterologous polynucleotide placed under transcriptional control of said promoter.
  • the present invention further provides recombinant expression vectors comprising an expression cassette of the invention, introduced into host cells and plants to produce transformed cells and transgenic plants.
  • chimaeric gene constructs containing hevein 5' flanking DNA linked to the uidA reporter gene was prepared and introduced into Hevea call us, and for some of the constructs, in model plants
  • hevein promoter in transformed cells and transgenic plants.
  • the promoter sequences of the hevein genes also acts as an inducible promoter regulated by wounds and pathogen infection.
  • Hevea brasiliensis ( illd. Ex Adr. Juss) is a tropical perennial euphorbiaceae originating from the /Amazon. It is from far the main crop exploited for the production of natural rubber in the world.
  • natural rubber occurs as a suspension of cis- 1, 4polyisoprene particles (from 0.01 to 15 ⁇ m in size) surrounded by a single membrane, in the latex, which is the cytoplasm of specialized cells called laticifers.
  • Genetic engineering of Hevea brasiliensis may be one way to improve the yield of natural rubber production, by over-expressing favorable genes of the latex metabolism or by improving the defenses of the tree. It is also an interesting tool for the production of recombinant proteins of industrial interest
  • the latex vessels formed by reticulated chains of contiguous anastomosed cells, are periodically emitted from the cambium towards the phloem, as concentric rings .
  • the latex vessels Upon tapping (excision of a thin layer of the trunk bark about 1 mm thick) , the latex vessels are severed and the latex flows out, until latex coagulation processes occur to plug the wound.
  • the expelled latex separates into three f actions: the rubber fraction, the C (cytosol) -serum and the "bottom" fraction composed of sedimentable organelles, mainly vacuolar elements known as lutoids .
  • the B-serum fluid found insides the lutoids, is very rich in proteins.
  • Hevein is a small single chain protein of 43 amino acids, unusually rich in cysteine and glycine
  • Lumpur pp 518-531, 1975
  • It is a monomer and has an apparent molecular weight of 9.5 kDa as determined by gel filtration and SDS-PAGE (VAN PARIJS et al . , Planta 183, 258- 264, 1991).
  • It is a chitin-binding protein that participates in the latex coagulation processes when released in the cytosol, by fixing the N-acetyl-D-glucosamine moiety of a receptor protein located at the surface of the rubber particles, therefore promoting their agglutination and the plugging of the tapping cut (GIDROL et al . , J. Biol. Chem. 269, 9278-9283, 1994) .
  • the mature hevein originates from a 204 aminoacids precursor protein which is matured by co- and post-translational processing, giving rise to 2 distinct domains (BROEKAERT et ai . , Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637, 1990; LEE et al . , J. Biol. Chem. 266(24), 15944- 15948, 1991) : the 43 aminoacids N-terminal chitin-binding domain (hevein) , specific of the lectins superfamily (VAN DAMME et al . , Plant Physiol.
  • prohevein can thus be classified as a PR-IV protein of class I, characterized by the presence of the chitin-binding N- terminal domain (VAN DAMME et al . , 1999, cited above).
  • VAN DAMME chitin-binding N- terminal domain
  • other evidences suggest that hevein plays a role in defense.
  • the hevein capacity to inhibit fungal growth was demonstrated in vi tro (VAN PARIJS et al . , 1991, cited above) .
  • hevein although poorly cleaved, displayed effective antifungal properties (LEE and RAIKHEL, Brazilian Journal of Medical & Biological Research 28, 743-750, 1995) .
  • Hevein is expressed at a high level in the latex compared to leaves, and is over- expressed by wounding, ethylene, and ABA (BROEKAERT et al . , Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637, 1990).
  • hevein is proposed to participate in the protection of the rubber tree trunk tissues, recurrently severed by the tapping process, by allowing the sealing of the tapping cut through the latex coagulation process and by preventing pathogenic infection. Only one nucleotide sequence encoding the full hevein precursor protein had been described so far: GenBank M36986 (BROEKAERT et al . , 1990, cited above).
  • GenBank AF327518 (DENG et al . )
  • GenBank AF287016 (AROKIARAJ and JONES) .
  • hevein precursors belong to a multigene family, that may be organized in two groups (group I and group II) based on nucleotide sequence homology. Further, the inventors have demonstrated that the 5' upstream region isolated from 3 different hevein genes, namely PHevl.l, representative of the hevein group I, and PHev2.1 (1824 bp) and PHev2.3
  • promoter sequences from the various hevein genes may be used as promoters in transformation programs, not only in Hevea but also in various other heterologous systems .
  • the first aspect of the present invention provides the shortest sequence shown by the inventors to be functional in Hevea as well as in rice is the PHevl.l sequence.
  • This sequence thus represents a promoter sequence able to drive the expression of a transgene in various plants, and constitutes a basis for the isolation of a longer promoter sequence, more readily usable as promoter in transformation programs.
  • the second aspect of the present invention provides the longest regulatory sequence tested by the inventors (PHev2.1) was demonstrated to be up-regulated by mechanical wounding in rice, with the capacity to respond systemically. It was also upregulated by pathogen infection.
  • polynucleotide selected among:
  • polynucleotide (g) is selected among:
  • polynucleotide selected among;
  • g' a polynucleotide having at least 90% identity with a polynucleotide (a' ) to (f' ) and having a promoter function in a plant cell.
  • said polynucleotide (g' ) comprises a sequence having at least 95% identity with a 97 bp sequence immediately upstream the ATG of a polynucleotide (a) to (f) .
  • polynucleotide (g) or (g') having at least 90% identity with said polynucleotide (c) is used as an inducible promoter regulable by wounds and pathogen infection.
  • the present invention also provides a recombinant expression cassette comprising a promoter consisting of a polynucleotide (a) to (g) , or preferably a polynucleotide (a' ) to (g' ) as defined above, and a heterologous polynucleotide placed under transcriptional control of said promoter.
  • heterologous polynucleotide refers herein to any polynucleotide other than an hevein coding sequence.
  • Said heterologous polynucleotide may consist for instance of a coding or of an antisense sequence of interest operably linked to said promoter, or of a cloning site for inserting said sequence of interest.
  • the present invention further provides recombinant expression vectors comprising an expression cassette of the invention. These expression vectors can be introduced into host cells and plants to produce transformed cells and transgenic plants, using methods known in the art.
  • Said transformed cells in particular plant cells, and transgenic plants are also part of the invention.
  • the present invention is suitable for use not only in Hevea and other plants of the euphorbeaceae family such as cassava or castor bean, but also in other dicotyledonous plants (as shown by the functionality of promoter sequences of the invention in the model plant Arabidopsis) , including in particular plants producing latex such as guayule, sunflower, lettuce, dendelion or papaya.
  • the present invention is also suitable for use in monocotyledonous plants, including in particular cereals, such as rice, maize, wheat, barley, or oats, and also including, in a non-limitative way, other plants such as banana, sugar cane or palm trees.
  • cereals such as rice, maize, wheat, barley, or oats
  • other plants such as banana, sugar cane or palm trees.
  • Figure 1A shows the synthetic oligonucleotide primers, starting with the primers T3, T7 and the reverse primer OR1, designed from the hevein cDNA sequence (Genbank accession M36986, position 58 to 83) for the isolation of the hevein gene promoter region PHevll, PHevl.2, PHev2.1 and PHev2.2.
  • Figure IB shows the sequences of all the oligonucleotide primers used for the isolation and cloning of HevP (PHev2.3) ;
  • Figure 2 shows the classification of the hevien genes and their sublcones .
  • Cloning I genomic sequences including the full transcribed region and 5' upstream sequences of hevien genes, cloned in the pBlueScript phagemid from lambda Zap II (stratagene) .
  • Cloning II Promoter regions isolated by PCT for Hevl.l, Hev 1.2 and Hev2.2, subcloning form the pBS-Hev clones into the pGe t- easy vector (Promega) and Hev2.3 (HevP) direct cloning by adaptator-anchored PCT in the Pcr2.1 TOPO vector (Invitrogen, USA).
  • Cloning III subcloning in a binary vector for transformation.
  • isolated promoter region cloned between the Hind III and Bgl II sited of the pCambial381Z vector (Ca bia) .
  • Ca bia pCambial381Z vector
  • HevG HevP
  • Figure 3 shows the nucleotide sequence of the hevein promoter region Phevl .1.
  • the transcription start site (A) and the translation initiation codon (ATG) are in bold letters .
  • Figure 4 shows the nucleotide sequence of the hevein promoter region Phevl .2.
  • the transcription start site (A) and the translation initiation codon (ATG) are in bold letters .
  • Figure 5 shows the nucleotide sequence of the hevein promoter region Phev2.1.
  • the transcription start site (A) and the translation initiation codon (ATG) are in bold letters .
  • Figure 6 shows the nucleotide sequence of the hevein promoter region Phev2.2.
  • the transcription start site (A) and the translation initiation codon (ATG) are in bold letters .
  • Figure 7 shows the nucleotide sequence of the hevein promoter region Phev2.3.
  • the transcription start site (A) and the translation initiation codon (ATG) are in bold letters .
  • Figure 8 shows the schematic diagram of the PCT- generated nested deletion fragments obtained from the hevein promoter region HevP (PHev2.3) and cloned in Ppgtv-Kan.
  • the promoter sequences were joined to the uidA reporter gene.
  • the 5' and 3' end points of the promoter sequences are numbered from the transcription start site of the hevein gene .
  • Figure 9 shows the fluorometric analysis of the GUS activity in transgenic rice plants (Tl) carrying a single copy of the uidA gene driven by PHev2.1, in response to mechanical wounding.
  • Figure 10 shows the results of fluorometric analysis of the GUS activity I transgenic rice plants (Tl) carrying a single copy of the uidA gene driven by PHev2.1, in response to fungal infection.
  • the present invention provides interesting tools for the genetic engineering of Hevea , with the purpose of improving existing genotypes for natural rubber production, or for molecular farming.
  • a wound-inducible promoter will guaranty a high transgene expression level in the exploited bark tissues, owing to the regular wound stress imposed by tapping.
  • This promoter will also be useful for increasing the expression of endogenous genes identified as limiting factors for the yield of latex production.
  • the invention also allows to optimize transgene expression in the latex cells; this is of particular importance for programs of molecular farming aiming at producing exogenous molecules of industrial interest in the latex.
  • the present invention can be used in Hevea as well as in other plant systems, for improving the defenses of plants against biotic and abiotic stresses.
  • the use of a promoter inducible by wounds and pathogen infection allows to over-express transgenes involved in defense, in situations such as aggressions by plant-eater insects or infection by microorganisms such as fungi.
  • EXAMPLE 1 CLONING OF HEVEIN-ENCODING GENOMIC SEQUENCES AND ISOLATION OF THE 5' UPSTREAM REGION
  • Hevea hevein is encoded by a small multigene family.
  • Four sequences (Hevl.l, Hevl.2, Hev2.1 and Hev2.2) are including the full coding sequence of the hevein precursor as well as the 5' upstream region.
  • One sequence includes the partial coding sequence and 5' upstream region of a fifth hevein gene (Hev2.3) .
  • a partial sequence of Hev2.3 is available in GenBank under the accession AF287016. Cloning of Heyl .1 , Heyl .2 , Hev2.1 and Hev2.2 by library screening and isolation of the 5' upstream region
  • Genomic DNA was extracted from young leaves of Hevea brasiliensis cultivar RRIM600, using the method described by DELLAPORTA et al . (Plant genetic Transformation and Gene Expression. A laboratory Manual, (Draper, J., Scott, R., Armirage, P. , Walden,R. eds), pp.214-216, Blackwell Scientific, London, UK, 1985) .
  • a genomic library was constructed by ligating
  • Hybridization was performed overnight at 65°C, in 5 x SSC (0.3 M NaCI, 30 M trisodium-citrate, pH 7), lOx Denhardt ' s reagent (0.2% ficoll, 0.2% PVP, 0.2% BSA), 7% SDS, 20 mM sodium phosphate buffer pH 7.2 and 100 ⁇ g ml -1 denatured salmon sperm DNA. Final washes were carried out at 65 °C, in 0.1 x SSC and 0.5% SDS.
  • the pBlue-Script SK(- ) phagemids containing the cloned genomic inserts were excised by co-infection with the ExAssist helper phage and transferred into E. coli XL1 blue.
  • the excised phagemids were extracted using Quiagen kits and mapping was performed using the enzymes Kpnl , EcoRI , Sa cl , Hindll l , Pstl and Xhol , in TA buffer (33 mM Tris acetate pH 7.9, 60 mM K + acetate, 10 mM Mg 2+ acetate, 0.5 mM DTT and 0.1 mg/ml of BSA, added extemporarily) , at 37°C.
  • the restriction fragments, separated by electrophoresis in 0.8% agarose gel were transferred onto nylon membranes and analyzed by Southern hybridization, to determine the orientation of the hevein gene inside the cloning vector.
  • Two probes were used successively: a cDNA fragment located near the 5' terminus of the cDNA (GenBank accession M36986, position 1-91), and the last 339 bp of the cDNA, including the whole 3' non coding region.
  • Four clones differing in restriction map were selected and sequenced on both strands using synthetic oligonucleotide primers, starting with the primers T3, T7 and the reverse primer OR1, designed from the hevein cDNA sequence (GenBank accession M36986, position 58 to 83) . These primers are shown in Figure 1A.
  • Phagemids pBS-Hevl.l and pBS-Hev2.1 were chosen for PCR amplification of the hevein gene 5' upstream region.
  • the forward primer SCH-S2 (Fig. 1A) was designed from the pBlue-Script SK phagemid vector, 12 bp from the T3 promoter.
  • the reverse primer SCH-R2 (Fig. 1A) was designed from the hevein gene upstream sequence PHev2.1, at position +27 to +48 from the transcription start site, 6 bp upstream the ATG described by BROEKAERT et al . (1990, cited above) as the translation initiation codon.
  • Amplification was performed in a Perkin Elmer thermocycler, in a 50 ⁇ l final volume, using 2 ng of Kpnl-linearized phagemid as matrix, and DNA polymerase mix and buffer from the "Expand High Fidelity PCR System" (Boehringer) , supplemented with MgCl 2 (3 mM) .
  • Amplification was performed over 35 cycles under the following conditions: denaturation at 94 °C for 45 sec, annealing at 50°C for 45 sec and elongation at 72°C for 2 min.
  • PCR fragments were purified by electrophoresis in 1.2% low melting agarose gel and eluted on affinity columns (NUCLEOSPIN Extract, Macherey-Nagel) and ligated in pGEM-T easy vector (Promega) .
  • the plasmid pCAMBIA 13812 was restricted using the enzymes Hin lll and Bgl II in buffer II (Boehringer Mannheim) , then dephosphorylated using alkaline phosphatase AP (Boehringer Mannheim) .
  • the plasmids pGEMT-PHevl.1 and pGEMT-PHev2.1 were restricted using the enzymes Hindlll and Bglll in buffer II (Boehringer Mannheim) .
  • the fragments corresponding to the promoter regions were isolated on 1.2% low melting agarose gel and purified using NUCLEOSPIN Extract columns (Macherey-Nagel) .
  • pCAMBIA-PHevl .1 and pCAMBIA-PHev2.1 were introduced into Agrobacteria (strain LBA 4404) by electroporation.
  • the constructs were re-extracted from the Agrobacterium using QIAGEN plasmid extraction system and checked by restriction profile analysis using the enzymes Xhol and Eco RV and by sequencing.
  • Hevl.l Hevl.2, Hev2.1 and Hev2.2
  • the four clones isolated include the full transcribed region of four different hevein genes, together with about 300 to 1.8 kb of their 5' upstream sequence.
  • the members of group I (Hev 1.1 and Hev 1.2) share about 96% identity while the members of group II (Hev2.1 and Hev2.2) share 99% identity.
  • the percentage of identity between members of the two different groups is about 86%.
  • High-molecular weight DNA was isolated from young leaves of RRIM 600 (Hevea brasiliensis) using the method as described by DELLAPORTA et al . (1985, cited above) .
  • Genomic mini-libraries were constructed using the Hevea genomic DNA by using the Universal GenomeWalkerTM Kit according to manufacturer's instructions (Clontech Laboratories, Inc, CA, USA) .
  • the mini-libraries were used as template DNA for PCR and nested PCR reactions for the isolation of hevein upstream sequences.
  • the oligonucleotide primers were synthesized by Operon (Operon Technologies Inc, USA) . The sequences of all the oligonucleotide primers used are listed in Figure IB.
  • DNA amplification was carried out on a thermal cycler, in a 50 ⁇ l volume, containing 5 ⁇ l of lOx Tth PCR reaction buffer (Clontech) , 2.2 ⁇ l of 25mM Mg(0Ac) 2 , 1 ⁇ l of
  • a secondary PCR reaction was performed using a 1:50 dilution of the primary PCR product.
  • DNA amplification was carried out on a thermal cycler, in a 50 ⁇ l volume, containing 5 ⁇ l of lOx Tth PCR reaction buffer (Clontech) , 2.2 ⁇ l of 25 mM Mg(0Ac) 2 , 1 ⁇ l of 10 ⁇ M of each primer (GSP2 hevein and AP2), 1 ⁇ l of lO ⁇ M each dNTPs, 1 ⁇ l of Advantage Polymerase Mix (2.5 units) (Clontech), 1 ⁇ l of the diluted
  • the amplified product (1.3 kb) was excised from the gel and purified.
  • the purified DNA fragment was cloned into TOPO Cloning TA vector pCR ® 2.1T0P0 ® (Invitrogen, USA) and transformed in E. Coli DHS ⁇ . Plasmid extractions were performed using the Qiagen Plasmid Mini Kit (Qiagen Inc, USA).
  • the 1.3 kb insert was released by restriction using the enzyme EcoR I, then sequenced (Strathclyde University, Department of Molecular Biology, United Kingdom) using as primers, M13R and M13F. Comparison of the promoter sequences of the hevein genes
  • the transcription start site was identified by primer extension from the clone PHev2.3, and deduced by sequence homology for the others. It is located 54 nt upstream the ATG codon described as the hevein mRNA translation start point.
  • the 5' upstream sequences of the five hevein genes are presented in Figures 3 (PHevl.l.), 4 (PHevl.2.), 5 (PHev2.1.), 6 (PHev2.2. ) and 7(PHev2.3.).
  • the transcription start site (A) and translation initiation codon (ATG) are in bold letters.
  • the 5' upstream regions of the 5 sequences PHevl.l, PHevl.2, PHev2.1, PHev2.2 and PHev2.3 were aligned together with a sequence released in the GenBank public database under the accession number AF327518.
  • All six promoter sequences are highly homologous (96% identity) over a 97 bp sequence immediately upstream the ATG (beginning at position -40 from the transcription start site) . Further upstream, the sequences diverge in two different groups, with 39-43% identity only between members of the 2 groups.
  • a high sequence homology is conserved among each group, with about 97% identity shared by members of group I (PHevl.l and PHevl.2) and 94-98% identity shared by members of group II (PHev2.1, PHev2.2, PHev2.3 and AF327518 ).
  • PHev2.3 although clearly belonging to group II, lacks a 30 bp domain located at position -173-143 from the transcription start site, when compared to PHev2.1, PHev2.2 and AF327518 .
  • EXAMPLE 2 FUNCTIONAL ANALYSIS IN HEVEA Deletion fragments from HevP (PHev2.3) .
  • the clone HevP (PHev2.3) was deleted to generate 4 overlapping fragments of decreasing length in the upstream region.
  • the amplified fragments were double digested with Xbal/ HindiII, then purified and ligated to pGPTV-KAN (BECKER et al . , Plant Moi. Biol. 20, 1195-1197, 1992) digested with Xbal/i ⁇ indlll and dephosphorylated.
  • the binary vector pGPTV-KAN contains unique cloning sites upstream of the uidA gene which allows the insertion of promoter fragments.
  • the hevein upstream fragments are fused to the uidA gene. The correct orientation and sequence of the fusions were verified.
  • FIG. 8 A schematic diagram of these constructs is represented in Figure 8.
  • the 5' and 3' end points of the promoter sequences are numbered from the transcription start of the Hevein gene.
  • pGPTV-KAN-1 (-275 to +55);
  • pGPTV-KAN-2 (- 389 to +55);
  • pGPTV-KAN-3 (-687 to +55) and
  • pGTV-KAN-4 (-919 to +55) .
  • Arrows indicate direction of transcription; R, right T-DNA border; L, left T-DNA border.
  • the 4 constructs generated (pGPTV-KAN-1; pGPTV- KAN-2; pGPTV-KAN-3; pGPTV-KAN-4 ) , together with pCAMBIA2301 as positive control, were transferred into Agrobacterium tumefaciens GV2260 by electroporation (SHEN and BRIAN, Nucleic Acid Research 17, 8395, 1989) .
  • the Hevea brasiliensis cultivar GL1 was transformed using the Agroba cterium tumefaciens-mediated protocol described by AROKIARAJ et al . (1998, cited above)
  • the transformed cells selected on kanamycin for about 2 months, were then screened for GUS expression by histochemical staining. GUS activity is indicated by a blue coloration after incubation with X-Gluc .
  • the calli carrying the various hevein promoter constructs and positive control pCAMBIA2301 displayed a blue colored surface, whereas this was not observed in the negative control sample. The same coloration was observed also in embryoids for all constructs except the negative control sample.
  • PHev2.1 containing respectively about 0.3 kb and 1.8 kb of the upstream sequence of Hevl .1 and Hev2.1 (hevein genes representative of group I and II respectively) fused to the uidA reporter gene, were introduced into inner integument- derived Hevea callus, by microprojectile bombardment- mediated transformation, to verify their functionality.
  • the cultivar RRIM 600 was transiently transformed by microprojectile bombardment of callus initiated from inner integument and maintained as described by CARRON et aJ. (Biotechnology in Agricultural and Forestry, (Bajaj, Y.P.S. ed) , pp 353-369, Springer Verlag, Berlin Heidelberg, 1995).
  • the particle gun was from BIORAD and the transformation procedure was as described by the manufacturer, with a 1100 psi rupture membrane. The distance between the rupture membrane and the macro-carrier was 6 cm. The distance between the macro-carrier and the callus was 6 cm.
  • the constructs pCAMBIA-PHevl .1 and pCAMBIA- PHev2.1 were introduced in rice, model plant for the monocots, via Agrobacterium-mediated transformation.
  • CCL liquid co-culture medium R2 medium from OHIRA et al . , Plant Cell Physiol. 14, 1113-1121, 1973; supplemented with 2.5 mg/1 2,4-D, 10 g/1 glucose, 100 ⁇ M acetosyringone, pH 5.2, then blotted dry and transferred onto solid co-culture medium (CCL with 7 g/1 agarose) .
  • the calli were transferred to R2S selection medium (R2 medium containing 30 g/1 glucose, 50 mg/1 hygromycin, 400 mg/1 cefotaxime, 100 mg/1 vancomycine, 7 g/1 agarose, pH ⁇ .O), at 27°C in the dark. After 2 weeks of selection, the calli were transferred to NBS medium (NB basic supplemented with 2.5 mg/1 2,4-D, 500 mg/1 proline, 500 mg/1 glutamine, 300 mg/1 casein hydrolysate, 50 mg/1 hygromycin, 400 mg/1 cefotaxime, 100 mg/1 vancomycine, 7 g/1 agarose, pH6.0).
  • R2S selection medium R2 medium containing 30 g/1 glucose, 50 mg/1 hygromycin, 400 mg/1 cefotaxime, 100 mg/1 vancomycine, 7 g/1 agarose, pH ⁇ .O
  • the resistant globular structures developed from the brownishing callus were separated and incubated for 10-15 days on fresh NBS medium, then placed on PRAG pre-regeneration medium (NB basic supplemented with 2 mg/1 BAP, 1 mg/1 NAA, 5 mg/1 ABA, 500 mg/1 proline, 500 mg/1 glutamine, 300 mg/1 casein nydrolysate, 50 mg/1 hygromycin, 100 mg/1 cefotaxime and 100 mg/1 vancomycine, 7 g/1 agarose, pH5.8), for one week.
  • PRAG pre-regeneration medium NB basic supplemented with 2 mg/1 BAP, 1 mg/1 NAA, 5 mg/1 ABA, 500 mg/1 proline, 500 mg/1 glutamine, 300 mg/1 casein nydrolysate, 50 mg/1 hygromycin, 100 mg/1 cefotaxime and 100 mg/1 vancomycine, 7 g/1 agarose, pH5.8
  • Creamy_white lobed calli were transferred to RN regeneration medium (NB basic supplemented with 3 mg/1 BAP, 0.5 mg/1 NAA, 30 g/1 glucose, 50 mg/1 hygromycin, 4.5 g/1 Phytagel, pH5.8), for 2 days in the dark then for 3 weeks with a 12 hours photoperiod.
  • Shoots regenerating from a resistant callus were subcultured in test tubes containing MS medium (MURASHIGE and SKOOG, Physiol. Plant 15, 473-497, 1962) with 50 g/1 glucose, 2.6 g/1 Phytagel, pH5.8. After 3 weeks, they were transferred to Jiffy peat pellets for 15 days, then to soil pots in the greenhouse. Only the hygromycin-resistant plants presenting a single T-DNA insertion were selected, by Southern blotting, for further analysis.
  • GUS activity was detected for some of the plants only (5 plants out of 9) , in wounded leaf tissues and in root vascular tissues. In the flowers and grains, the coloration was limited to the lemma and palea.
  • Proteins from the supernatant were quantified (Bradford, 1976) using a Multiscan RC (Labsystems) spectrophotometer, coupled to the software Genesis version 2.00 (Labsystems).
  • the fluorescence of 4-MU (4-methylumbelliferone) generated after cleavage of the 4- MUG (4-methylumbelliferyl ⁇ -D-glucuronic acid) substrate by the GUS enzyme was measured at 460 nm using the fluorimeter "Fluoroscan II version 6.3 (Labsystems)" coupled to the software Genesis version 2.00.
  • Tl population The growth conditions were strictly controlled: 25°C, 75% humidity and 11 h 30 min under artificial light (400 ⁇ molE.m-2. s _1 ) . Seeds were collected and dried at 37 °C for 3 days, then kept at room temperature.
  • the regulation of the hevein promoters by mechanical wounding was verified by fluorimetrical analysis. Mechanical wounding was performed by pricking with needles the whole surface of the last fully developed leaf. Four different transgenic lines bearing the PHev2.1 promoter region were analyzed. For each line, 7 batches of 3 Tl plants were used. For one batch (To) , the last fully developed leaf was collected, before any wounding. Five other batches (Ti, T 3 , T 6 , T 10 and T 24 ) were submitted to mechanical wounding by pricking with needles the whole limb surface of the last fully developed leaf, then the wounded leaves were collected respectively 1, 3, 6, 10 and 24 hours after wounding.
  • the GUS activity value represented is the average value from 4 different transgenic lines, each line being represented by the average value from 3 Tl progenies .
  • All four lines bearing the PHev2.1 promoter region displayed a significant overexpression of the uidA gene in response to wounding, with maximum amplitude observed 10 hours after wounding (amplification factor 1.4 to 1.6). This induction was statistically highly significant (P ⁇ 0.01) as evaluated by Fisher test (LSD). No significant change was observed for the plants bearing the CaMV 35S promoter. The level of activity of the wound-stimulated PHev2.1 promoter is thus very close (80%) to the level displayed by the 35S promoter in similar conditions. Moreover, the PHev2.1 promoter appeared to respond also systemically to wounding. The amplification factor measured on intact leaves framed by wounded leaves 10 hours after wounding was about 1.2 in average.
  • Arabidopsis thaliana (ecotype ColO) was transformed in planta as described by Clough and Bent (1998). Seeds were collected and stored at 4°C, at least for one week, for vernalisation. Selection of the transgenic events was performed as follows: seed were sterilized for 20 min in a mixture of 30% sodium hypochlorite, absolute ethanol and water (1/4/3; v/v/v) , rinsed three times with absolute ethanol and dried for 2 hours. They were then sown and grown in vi tro on MS/2 medium supplemented with 30 ⁇ g.ml "1 hygromycin. The hygromycin resistant seedlings (transformed events of first generation, or TO) were transferred to the green house after development of the first leaves. Transformed lines of second generation (Tl) were similarly selected on hygromycin.

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Abstract

La présente invention se rapporte à la mise en évidence de séquences d'ADN provenant de l'espèce Hevea brasiliensis contenant la région de promoteur et la région régulatrice des gènes d'hévéine. Les séquences de type promoteur des gènes d'hévéine agissent également comme un promoteur inductible régulé par les plaies et les infections dues à un agent pathogène.
PCT/IB2004/001904 2003-05-16 2004-05-13 Sequences de type promoteur provenant des genes d'heveine de l'espece hevea brasiliensis WO2004101614A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105886528A (zh) * 2016-01-28 2016-08-24 中国热带农业科学院橡胶研究所 一种用乳管特异性启动子获得橡胶树转基因植株的方法
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
CN113025616A (zh) * 2021-04-21 2021-06-25 中国热带农业科学院橡胶研究所 一种橡胶树泛素基因启动子proHbUBI2及其克隆与应用

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US6083687A (en) * 1990-09-24 2000-07-04 Board Of Trustees Operating Michigan State University cDNA encoding a polypeptide including a hev ein sequence

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BROEKAERT W ET AL: "WOUND-INDUCED ACCUMULATION OF MESSENGER RNA CONTAINING A HEVEIN SEQUENCE IN LATICIFERS OF RUBBER TREE HEVEA-BRASILIENSIS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 87, no. 19, 1990, pages 7633 - 7637, XP002297173, ISSN: 0027-8424 *
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20160244774A1 (en) * 2015-02-23 2016-08-25 Sumitomo Rubber Industries, Ltd. Vector comprising specific promoter and gene encoding specific protein, transgenic plant into which the vector has been introduced, and method for improving polyisoprenoid production by introducing the vector into plant
JP2016154458A (ja) * 2015-02-23 2016-09-01 住友ゴム工業株式会社 特定のプロモーター及び特定の蛋白質をコードする遺伝子を含むベクター、該ベクターが導入された形質転換植物、並びに、該ベクターを植物に導入することにより、ポリイソプレノイドの生産量を向上させる方法
CN105886528A (zh) * 2016-01-28 2016-08-24 中国热带农业科学院橡胶研究所 一种用乳管特异性启动子获得橡胶树转基因植株的方法
CN105886528B (zh) * 2016-01-28 2020-06-23 中国热带农业科学院橡胶研究所 一种用乳管特异性启动子获得橡胶树转基因植株的方法
CN113025616A (zh) * 2021-04-21 2021-06-25 中国热带农业科学院橡胶研究所 一种橡胶树泛素基因启动子proHbUBI2及其克隆与应用
CN113025616B (zh) * 2021-04-21 2023-05-02 中国热带农业科学院橡胶研究所 一种橡胶树泛素基因启动子proHbUBI2及其克隆与应用

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