WO2000037661A1 - MODIFICATION GENETIQUE DE $i(COMPOSITAE) - Google Patents

MODIFICATION GENETIQUE DE $i(COMPOSITAE) Download PDF

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Publication number
WO2000037661A1
WO2000037661A1 PCT/GB1999/004317 GB9904317W WO0037661A1 WO 2000037661 A1 WO2000037661 A1 WO 2000037661A1 GB 9904317 W GB9904317 W GB 9904317W WO 0037661 A1 WO0037661 A1 WO 0037661A1
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WIPO (PCT)
Prior art keywords
plant
gene
rna
act2
express
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PCT/GB1999/004317
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English (en)
Inventor
Cornelis M. P. Van Dun
Frank M. A. Schepers
Jan H. Pertijs
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Advanta Technology Limited
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Publication date
Application filed by Advanta Technology Limited filed Critical Advanta Technology Limited
Priority to AU18738/00A priority Critical patent/AU1873800A/en
Publication of WO2000037661A1 publication Critical patent/WO2000037661A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • 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

Definitions

  • the present invention relates to the genetic modification of plants of the family Compositae, in particular lettuce (Lactuca sativa) and sunflower (He anthus annuus)
  • heterologous genes allows the plant to produce heterologous proteins with functions that the plant does not normally possess
  • introduction of the gene for production of the Bacillus thunngiensis (Bt) insecticidal protein renders the plant toxic to insects and protects it against insect attack
  • genes may be introduced which protect against the attack of fungal and viral diseases
  • genetic modification to control the expression of homologous genes which the plant already possesses
  • down-regulation may be induced by means such as anti- sense technology, in which an inverted copy of the homologous gene is inserted in the plant Expression of the inverted gene produces antisense RNA, which inhibits the expression of the natural gene In this way, for example, ripening of fruit such as
  • a method of producing a genetically-modified Compositae plant which comprises transforming the plant with a heterologous DNA construct including a DNA sequence adapted to express RNA in the plant under the control of the act ⁇ n2 (ACT2) gene promoter
  • ACT2 act ⁇ n2
  • Compositae plant cells comprising a heterologous DNA construct including a DNA sequence adapted to express RNA in the plant under the control of the act ⁇ n2 (ACT2) gene promoter
  • Compositae in particular lettuce and sunflower, plants comprising such cells
  • the promoter of the act ⁇ n2 (ACT2) gene derived from Arabidopsis has been shown to drive the beta-glucuronidase (GUS) gene in transgenic Arabidopsis plants to relatively high levels in vegetative tissues In the inflorescence a developmental factor seemed to be involved causing a more differentiated expression pattern This is reported by An, Meagher et a/ (1996), The Plant Journal 10, 107-121 the same reference gives the DNA sequence
  • Figure 1 is a map of a construct pVDH380 comprising the promoter of the act ⁇ n2
  • ACT2 gene derived from Arabidopsis arranged to drive the beta-glucuronidase (GUS) gene
  • Figure 2 is a map of a construct pVDH641 comprising the promoter of the act ⁇ n2 (ACT2) gene derived from Arabidopsis arranged to drive the OXOX gene of wheat
  • Figure 3 gives the DNA sequence of the act ⁇ n2 (ACT2) promoter derived from
  • Actin is a fundamental cytoskeletal component essential to nearly all eukaryotic cells, in which it forms microfilament structures
  • the actin 2 gene promoter is a constitutive promoter to be found in most plants
  • the DNA sequence which expresses RNA may be of two mam kinds either a sequence which expresses mRNA which is translated into protein, or a sequence which produces RNA which is not translated into protein, but which interacts with the biochemistry of the plant cell in another way, for example by inhibiting gene expression
  • a preferred use of the present invention is to promote the expression of heterologous genes However it may also be used to up-regulate or down-regulate the expression of homologous genes
  • heterologous genes may be used DNA sequences coding for insecticidal proteins (for example the Bt protein) or fungicidal or antiviral proteins ACT2 can be used to drive oxox (the oxalate oxidase gene), giving sclerotinia resistance, or other genes like early or late flowering genes (for example ATH1 ), herbicide resistance genes, insect tolerance genes (aphid resistance in lettuce), virus resistance genes (eg lettuce mosaic virus, LMV), nitrate reductase to lower nitrate content, and genes for increased shelf life Many traits desirable in lettuce and sunflower can be exploited using this promoter
  • a DNA construct for use in the invention may be an "antisense” construct generating “antisense” RNA or a “sense” construct (encoding at least part of the functional protein) generating “sense” RNA
  • Antisense RNA is an RNA sequence which is complementary to a sequence of bases in the corresponding mRNA complementary in the sense that each base (or the majority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5' to 3' sense.
  • Such antisense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged to generate a transcript with at least part of its sequence complementary to at least part of the coding strand of the relevant gene (or of a DNA sequence showing substantial homology therewith).
  • Sense RNA is an RNA sequence which is substantially homologous to at least part of the corresponding mRNA sequence.
  • Such sense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged in the normal orientation so as to generate a transcript with a sequence identical to at least part of the coding strand of the relevant gene (or of a DNA sequence showing substantial homology therewith).
  • Suitable sense constructs may be used to inhibit gene expression (as described in International Patent Publication WO91/08299) or a sense construct encoding and expressing a homologous functional protein may be transformed into the plant to over-express the protein.
  • DNA constructs for use in the invention to inhibit gene expression may comprise a base sequence at least 10 bases (preferably at least 35 bases) in length for transcription into RNA. There is no theoretical upper limit to the base sequence - it may be as long as the relevant mRNA produced by the cell - but for convenience it will generally be found suitable to use sequences between 100 and 1000 bases in length.
  • a suitable cDNA or genomic DNA or synthetic polynucleotide may be used as a source of the DNA base sequence for transcription.
  • FIG. 1 Lettuce was transformed with various constructs comprising the act2 gene promoter
  • the act2/GUS construct shown in Figure 1 kindly provided by courtesy of Dr R B Meagher, Dept of Genetics, University of Georgia, Athens, GA 30602, USA, and by us termed pVDH380 ( Figure 1 ), was used to transform lettuce, in order to evaluate the expression pattern in primary transformants as well as the stability in consecutive generations
  • the construct pVDH380 was used to make a construct pVDH641 ( Figure 2) containing the act2 promoter linked to the oxalate oxidase (OXOX) gene of wheat this construct was used in transient expression studies in both lettuce and sunflower
  • the binary vector pVDH380 of Figure 1 contains the NPTII gene as a selectable marker in addition to the ACT2/GUS gene
  • the act2 promoter sequence includes the first 19 codons of the act2 gene as well as the first exon-intron combination Vector pVDH380 was used directly to transform lettuce (variety "Evola"), following the Ti plasmid method given in Curtis et al (1994), J Exp Bot 45, 1441-1449
  • act2/GUS construct displays a relatively strong, predominantly constitutive expression pattern in transgenic lettuce at the TO level
  • pVDH641 Figure 2
  • pVDH380 Figure 1 which is identical with the plasmid ACT2/GUS described by An et al., cited above.
  • Figure 1 shows a physical map of the construct pVDH380.
  • 'LB' indicates the left border
  • 'RB' indicates the right border
  • 'Pnos' indicates the nopaline synthase promoter
  • Tnos' indicates the nopaline synthase terminator
  • 'NPTII' indicates the neomycin phosphotransferase II gene
  • 'pACT2' indicates the Actin 2 promoter
  • 'GUS' indicates the beta-glucuronidase gene
  • 'KanR' indicates the bacterial kanamycin resistance gene.
  • the ACT2 promoter was recloned from vector pVDH380 after amplification by PCR.
  • Figure 3 shows the nucleotide sequence of the Actin 2 promoter region.
  • the sequence corresponding to the forward primer (bold, Gothic typeface) as well as to the complementary sequence (bold underlined) of the backward primer are indicated
  • the start codon of the Actin 2 gene, ATG is given in bold capitals
  • the composition of the forward and backward primers are given We used a p merset consisting of primer 1 (5'-GC AAGCTT ATT ATG ATC TCA AAT ACA TTG-3') and primer 2 (5'-GC GGATCC TTT ATG AGC TGC AAA CAC AC-3')
  • Primer 1 contains after the first two nucleotides a Hindlll restriction recognition site and subsequently a nucleotide sequence identical to the nucleotide sequence located from position 1358 to position 1379 upstream from the ATG-start codon (see Figure 3)
  • Primer 2 contains after the first two nucleotides a BamHI restriction recognition site and subsequently 20 nu
  • OxOx activity can be measured in a histochemical assay using oxalate which is converted by the enzyme into a purple dye
  • the act2/OXOX fusion showed good levels of OxOx activity in transient assays, using both lettuce and sunflower explants, confirming the functionality of the construct.

Abstract

La présente invention concerne un procédé permettant de produire une plante compositae modifiée génétiquement. Cette plante est transformée avec une construction d'ADN hétérologue comprenant une séquence d'ADN adaptée de façon à exprimer l'ARN dans la plante sous le contrôle du promoteur du gène actine2 (ACT2).
PCT/GB1999/004317 1998-12-21 1999-12-16 MODIFICATION GENETIQUE DE $i(COMPOSITAE) WO2000037661A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU18738/00A AU1873800A (en) 1998-12-21 1999-12-16 Genetic modification of (compositae)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9828201.5A GB9828201D0 (en) 1998-12-21 1998-12-21 Genetic modification of compositae
GB9828201.5 1998-12-21

Publications (1)

Publication Number Publication Date
WO2000037661A1 true WO2000037661A1 (fr) 2000-06-29

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GB (1) GB9828201D0 (fr)
WO (1) WO2000037661A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000070067A1 (fr) * 1999-05-14 2000-11-23 Dekalb Genetics Corporation Promoteur actine 2 de riz et intron, procedes d'utilisation associes
WO2001044457A3 (fr) * 1999-12-16 2002-01-10 Monsanto Technology Llc Nouvelles constructions d'expressions vegetales
US8877916B2 (en) 2000-04-26 2014-11-04 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992014824A1 (fr) * 1991-02-25 1992-09-03 Imperial Chemical Industries Plc Oxalate oxydase nouvellement caracterisee et utilisations de celle-ci
WO1997032011A1 (fr) * 1996-02-28 1997-09-04 Novartis Ag Molecules d'adn codant pour la protoporphyrinogene-oxydase vegetale et mutants de cette enzyme resistants aux inhibiteurs
WO2000020571A2 (fr) * 1998-10-06 2000-04-13 Pioneer Hi-Bred International, Inc. Promoteurs du maïs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992014824A1 (fr) * 1991-02-25 1992-09-03 Imperial Chemical Industries Plc Oxalate oxydase nouvellement caracterisee et utilisations de celle-ci
WO1997032011A1 (fr) * 1996-02-28 1997-09-04 Novartis Ag Molecules d'adn codant pour la protoporphyrinogene-oxydase vegetale et mutants de cette enzyme resistants aux inhibiteurs
WO2000020571A2 (fr) * 1998-10-06 2000-04-13 Pioneer Hi-Bred International, Inc. Promoteurs du maïs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AN YONG-QIANG ET AL: "Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues.", PLANT JOURNAL 1996, vol. 10, no. 1, 1996, pages 107 - 121, XP000876842, ISSN: 0960-7412 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6429357B1 (en) 1999-05-14 2002-08-06 Dekalb Genetics Corp. Rice actin 2 promoter and intron and methods for use thereof
WO2000070067A1 (fr) * 1999-05-14 2000-11-23 Dekalb Genetics Corporation Promoteur actine 2 de riz et intron, procedes d'utilisation associes
US7141722B2 (en) 1999-12-16 2006-11-28 Monsanto Technology Llc Glyphosate resistant plants using hybrid promoter constructs
US6919495B2 (en) 1999-12-16 2005-07-19 Monsanto Technology Llc Chimeric cauliflower mosaic virus 35S-arabidopsis actin 8 promoters and methods of using them
US6949696B2 (en) 1999-12-16 2005-09-27 Monsanto Technology Chimeric figwort mosaic virus-elongation factor 1 α promoters and methods of using them
US7112725B2 (en) 1999-12-16 2006-09-26 Monsanto Technology Llc Plants having high plant map values
WO2001044457A3 (fr) * 1999-12-16 2002-01-10 Monsanto Technology Llc Nouvelles constructions d'expressions vegetales
CN100425701C (zh) * 1999-12-16 2008-10-15 孟山都技术有限公司 新型植物表达构建物
US8822666B2 (en) 1999-12-16 2014-09-02 Monsanto Technology Llc Hybrid caulimovirus promoters and constructs thereof
US8987434B2 (en) 1999-12-16 2015-03-24 Monsanto Technology Llc Hybrid caulimovirus promoters and constructs thereof
US9347069B2 (en) 1999-12-16 2016-05-24 Monsanto Technology Llc Hybrid caulimovirus promoters and constructs thereof
US9856491B2 (en) 1999-12-16 2018-01-02 Monsanto Technology Llc Hybrid caulimovirus promoters and constructs thereof
US8877916B2 (en) 2000-04-26 2014-11-04 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof

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AU1873800A (en) 2000-07-12
GB9828201D0 (en) 1999-02-17

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