EP0729512A1 - Adn, produits de recombinaison d'adn, cellules et plantes derivees desdits produits - Google Patents

Adn, produits de recombinaison d'adn, cellules et plantes derivees desdits produits

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Publication number
EP0729512A1
EP0729512A1 EP95900873A EP95900873A EP0729512A1 EP 0729512 A1 EP0729512 A1 EP 0729512A1 EP 95900873 A EP95900873 A EP 95900873A EP 95900873 A EP95900873 A EP 95900873A EP 0729512 A1 EP0729512 A1 EP 0729512A1
Authority
EP
European Patent Office
Prior art keywords
plants
sam
dna
decarboxylase
plant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95900873A
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German (de)
English (en)
Inventor
Donald Grierson
Rupert George 44 Packington Hill FRAY
Andrew David 21 Packington Hill WALLACE
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Syngenta Ltd
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Zeneca Ltd
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Publication date
Application filed by Zeneca Ltd filed Critical Zeneca Ltd
Publication of EP0729512A1 publication Critical patent/EP0729512A1/fr
Withdrawn legal-status Critical Current

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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • 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
    • C12N9/88—Lyases (4.)

Definitions

  • This invention relates to DNA constructs, plant cells containing the constructs and plants derived therefrom. In particular it relates to the modification of plant gene expression causing modification of senescence or ripening characteristics.
  • the modification of plant gene expression has been achieved by several methods.
  • the molecular biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal expression of a particular gene.
  • the expression of either specific antisense RNA or partial (truncated) sense RNA has been utilised to reduce the expression of various target genes in plants (as reviewed by Bird and Ray, 1991, Biotechnology and Genetic Engineering Reviews 9:207-227).
  • These techniques involve the incorporation of a synthetic gene into the genome of the plant, the synthetic gene being designed to express either antisense or sense RNA.
  • RNA containing the complete coding region of the target gene may be incorporated into the genome of the plant to "over-express" the gene product.
  • Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences .
  • MTA 5-methylthioadenosine
  • MTR 5-methylthioribose
  • MTR-IP 5-methylthioribose-1-phosphate
  • KMB 2-keto-4-methythiobutyrate
  • Met methionine
  • SAM S-adenosyl methionine
  • dSAM decarboxylated S-adenosyl methionine
  • ACC 1-aminocyclopropane-l-carboxylic acid
  • MACC malonyl ACC.
  • SAM S-adenosyl methionine
  • SAM-decarboxylase genes have been isolated from animal sources. Recently, a cDNA clone isolated from potato Solanum tuberosum was found to be homologous to a human SAM-decarboxylase gene. The potato cDNA clone was designated TUB13 (Taylor et al, 1992, Plant Molecular Biology, 20:641-651; EMBL/GenBank accession number Z11680) . Southern analysis also showed homologous sequences in the potato Solanum brevidens and in the tomato Lvcopersicon esculentum, although these sequences were not elucidated.
  • a method to modify plant characteristics comprising modification of the activity of S-adenosyl methionine decarboxylase.
  • the method may be used to modify plant senescence characteristics and/or fruit ripening characteristics.
  • SAM-decarboxylase S-adenosyl methionine decarboxylase
  • the levels of SAM-decarboxylase may be either reduced or increased depending on the characteristics desired for the modified plant part (fruit, leaf, flower, etc) .
  • Antisense or “partial sense” or other techniques -may be used to reduce the expression of SAM-decarboxylase.
  • the levels of SAM-decarboxylase may also be increased; for example, by incorporation of additional SAM-decarboxylase genes.
  • the additional genes may be designed to give either the same or different spatial and temporal patterns of expression in the fruit.
  • SAM decarboxylase Over-expression of SAM decarboxylase will compete with the conversion of SAM to ethylene, thus reducing ethylene production.
  • the alteration in ethylene levels will modify ripening and senescence processes. Further, the resulting additional polyamine accumulation may antagonise ethylene-stimulated ripening and senescence of flowers, leaves etc.
  • Increased activity of SAM decarboxylase may be used to modify various aspects of plant quality, including: a. Increased fruit firmness; b. Improved resistance of fruit to mechanical damage during harvest and subsequent handling due to slowing of the ripening and over-ripening processes; c. Improved texture of ripe fruit; d. Improved resistance to fruit diseases; e. Improved viscosity of processed fruit; f.
  • Inhibition of SAM decarboxylase may result in increased ethylene production, enhancing senescence and/or ripening processes.
  • Reduced activity of SAM decarboxylase may be used to modify various aspects of plant quality, including: a. Modified fruit texture; b. Modified processing properties; c. Effects of increased ethylene synthesis; d. Altered flavour.
  • cDNA of this gene has been cloned and characterised and may be used to modify the senescence characteristics of plant parts and/or the ripening characteristics of fruit.
  • a DNA sequence encoding a SAM-decarboxylase may be derived from cDNA, from genomic DNA or may be synthesised ab initio.
  • a cDNA clone encoding a SAM-decarboxylase has been obtained from a tomato cDNA library.
  • the clone is hereinafter called GUP16.
  • the nucleotide sequence of the SAM-decarboxylase cDNA (clone GUP16) is given as SEQ ID NO 1.
  • the clone GUP16 (in E coli strain XLl-BLue) was deposited at The National Collections of Industrial and Marine Bacteria (23 St Machar Drive, Aberdeen, Scotland, AB2 1RY) under the terms of the Budapest Treaty on 8 November 1993 under the accession number NCIMB 40596.
  • An alternative source of the DNA sequence is a suitable gene encoding a SAM-decarboxylase.
  • This gene may differ from the corresponding cDNA in that introns may be present. The introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out) .
  • Oligonucleotide probes or the cDNA clone may be used to isolate the actual SAM-decarboxylase gene(s) by screening genomic DNA libraries. Such genomic clones may include control sequences operating in the plant genome.
  • promoter sequences which may be used to drive expression of the enzymes or any other protein. These promoters may be particularly responsive to certain developmental events (such as senescence or ripening) and environmental conditions.
  • SAM-decarboxylase gene promoters may be used to drive expression of any target gene.
  • a SAM-decarboxylase DNA sequence may be isolated from cDNA or genomic DNA libraries of any suitable plant species using oligonucleotide probes based on the GUP16 sequence. Bacterial, fungal, algal, yeast or eukaryotic DNA libraries may also be probed for SAM-decarboxylase sequences.
  • a SAM-decarboxylase DNA sequence is any sequence which cross-hybridises with SEQ ID NO 1, preferably having at least 60% homology with SEQ ID NO 1.
  • a SAM-decarboxylase DNA sequence may encode a protein which is homologous to the predicted gene product encoded by SEQ ID NO 1.
  • a further way of obtaining a SAM-decarboxylase DNA sequence is to synthesise it ab initio from the appropriate bases, for example using the appropriate cDNA sequence as a guide.
  • SAM-decarboxylase sequence may be incorporated into DNA constructs suitable for plant transformation. These DNA constructs may then be used to modify SAM-decarboxylase gene expression in plants. "Antisense” or “partial sense” or other techniques may be used to reduce SAM-decarboxylase gene expression in plant tissue (down-regulation) . The levels of expression may also be increased (up-regulation) ; for example, by incorporation of additional SAM-decarboxylase genes. The additional genes may be designed to give either the same or different spatial and temporal patterns of expression in the plant. According to a further aspect of the invention there is provided a DNA construct comprising some or all of a SAM-decarboxylase DNA sequence under the control of a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.
  • the senescence and/or ripening characteristics and related characteristics of plant parts may be modified by transformation with a DNA construct according to the invention.
  • the invention also provides plant cells containing such constructs; plants derived therefrom having modified SAM-decarboxylase gene expression; and seeds of such plants.
  • a DNA construct according to the invention may be an "antisense” construct generating “antisense” RNA or a “sense” construct (encoding at least part of the functional enzyme) 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 the functional enzyme may be transformed into the plant to over-express the enzyme.
  • DNA constructs according to the invention may comprise a base sequence at least 10 bases (preferably at least 35 bases) in length for transcription into RNA.
  • base sequence at least 10 bases (preferably at least 35 bases) in length for transcription into RNA.
  • sequences between 100 and 1000 bases in length but there is no theoretical upper limit to the base sequence - it may be as long as the relevant mRNA produced by the cell (for example, the GUP16 cDNA sequence is 1445 base pairs in length) .
  • the preparation of such constructs is described in more detail below.
  • a suitable cDNA or genomic DNA or synthetic polynucleotide may be used as a source of the DNA base sequence for transcription.
  • the isolation of suitable SAM-decarboxylase sequences is described above. Sequences coding for the whole, or substantially the whole, of the enzyme may thus be obtained. Suitable lengths of this DNA sequence may be cut out for use by means of restriction enzymes.
  • genomic DNA As the source of a partial base sequence for transcription it is possible to use either intron or exon regions or a combination of both.
  • the cDNA sequence as found in the enzyme cDNA or the gene sequence as found in the chromosome of the plant may be used.
  • Recombinant DNA constructs may be made using standard techniques.
  • the DNA sequence for transcription may be obtained by treating a vector containing said sequence with restriction enzymes to cut out the appropriate segment.
  • the DNA sequence for transcription may also be generated by annealing and ligating synthetic oligonucleotides or by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to give suitable restriction sites at each end.
  • the DNA sequence is then cloned into a vector containing upstream promoter and downstream terminator sequences. If antisense DNA is required, the cloning is carried out so that the cut DNA sequence is inverted with respect to its orientation in the strand from which it was cut.
  • RNA in a construct expressing antisense RNA the strand that was formerly the template strand becomes the coding strand, and vice versa.
  • the construct will thus encode RNA in a base sequence which is complementary to part or all of the sequence of the enzyme mRNA.
  • the two RNA strands are complementary not only in their base sequence but also in their orientations (5' to 3').
  • RNA In a construct expressing sense RNA, the template and coding strands retain the assignments and orientations of the original plant gene. Constructs expressing sense RNA encode RNA with a base sequence which is homologous to part or all of the sequence of the mRNA. In constructs which express the functional enzyme, the whole of the coding region of the gene is linked to transcriptional control sequences capable of expression in plants.
  • constructs according to the present invention may be made as follows.
  • a suitable vector containing the desired base sequence for transcription such as the GUP16 cDNA clone
  • restriction enzymes to cut the sequence out.
  • the DNA strand so obtained is cloned (if desired, in reverse orientation) into a second vector containing the desired promoter sequence and the desired terminator sequence.
  • Suitable promoters include the 35S cauliflower mosaic virus promoter and the tomato polygalacturonase gene promoter sequence (Bird et al, 1988, Plant Molecular Biology, 11:651-662) or other developmentally regulated fruit promoters.
  • Suitable terminator sequences include that of the Agrobacterium tumefaciens nopaline synthase gene (the nos 3' end) .
  • the transcriptional initiation region may be derived from any plant-operative promoter.
  • the transcriptional initiation region may be positioned for transcription of a DNA sequence encoding RNA which is complementary to a substantial run of bases in a mRNA encoding the SAM-decarboxylase enzyme (making the DNA construct a full or partial antisense construct) .
  • the transcriptional initiation region (or promoter) operative in plants may be a constitutive promoter (such as the 35S cauliflower mosaic virus promoter) or an inducible or developmentally regulated promoter (such as fruit-specific promoters) , as circumstances require. For example, it may be desirable to modify SAM-decarboxylase activity only during fruit development and/or ripening.
  • a constitutive promoter will tend to affect enzyme levels and functions in all parts of the plant, while use of a tissue specific promoter allows more selective control of gene expression and affected functions. Thus in applying the invention it may be found convenient to use a promoter that will give expression during fruit development and/or ripening. Thus the antisense or sense RNA is only produced in the organ in which its action is required.
  • Fruit development and/or ripening-specific promoters that could be used include the ripening-enhanced polygalacturonase promoter (International Patent Publication Number WO92/08798) , the E8 promoter (Diekman &. Fischer, 1988, EMBO, 7:3315-3320) and the fruit specific 2A11 promoter (Pear et al, 1989, Plant Molecular Biology, 13:639-651) .
  • the DNA constructs of the invention may be inserted into plants to regulate the expression of SAM-decarboxylase genes and the production of SAM-decarboxylase enzymes, resulting in modification of ethylene and/or polyamine metabolism and consequent modification of plant characteristics (in particular senescence and/or fruit-ripening characteristics) .
  • the production of the SAM-decarboxylase may be increased, or reduced, either throughout or at particular stages in the life of the plant.
  • production of the enzyme is enhanced only by constructs which express RNA homologous to the substantially complete endogenous enzyme mRNAs.
  • Full-length sense constructs may also inhibit enzyme expression. Constructs containing an incomplete DNA sequence shorter than that corresponding to the complete gene generally inhibit the expression of the gene and production of the enzymes, whether they are arranged to express sense or antisense RNA. Full-length antisense constructs also inhibit gene expression.
  • a DNA construct of the invention is transformed into a target plant cell.
  • the target plant cell may be part of a whole plant or may be an isolated cell or part of a tissue which may be regenerated into a whole plant.
  • the target plant cell may be selected from any monocotyledonous or dicotyledonous plant species. Plants may be derived from the transformed plant cell by regeneration of transformants and by production of successive generations of the transformants' progeny.
  • Constructs according to the invention may be used to transform any plant using any suitable transformation technique to make plants according to the invention.
  • Both monocotyledonous and dicotyledonous plant cells may be transformed in various ways known to the art. In many cases such plant cells (particularly when they are cells of dicotyledonous plants) may be cultured to regenerate whole plants which subsequently reproduce to give successive generations of genetically modified plants.
  • Any suitable method of plant transformation may be used.
  • dicotyledonous plants such as tomato and melon may be transformed by A ⁇ robacterium Ti plasmid technology, such as described by Bevan (1984, Nucleic Acid Research, 12:8711-8721) or Fillatti et al (Biotechnology, July 1987, 5:726-730). Such transformed plants may be reproduced sexually, or by cell or tissue culture.
  • Examples of genetically modified plants according to the present invention include all flowering plants, all vegetables (including tubers such as radishes, turnips and potatoes) and all fruit-bearing plants (such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, peppers, chillies, paprika) .
  • Other plants that may be modified by the process of the invention include cereals such as maize (corn) , wheat, barley and rice.
  • SAM-decarboxylase constructs allows modification of SAM-decarboxylase enzyme activity and thus provides a method for modification of plant characteristics, particularly senescence and fruit-ripening.
  • the overall level of SAM-decarboxylase activity and the relative activities of other enzymes affect plant development and thus determine certain characteristics of the plant parts. Modification of SAM-decarboxylase activity can therefore be used to modify various aspects of plant quality.
  • the activity levels of the enzyme may be either increased or reduced during development depending on the characteristics desired for the modified plant.
  • the SAM-decarboxylase gene may also be expressed in cells, tissues and organisms that do not normally produce the enzyme.
  • SAM-decarboxylase gene expression may be modified to a greater or lesser extent by controlling the degree of the sense or antisense mRNA production in the plant cells. This may be done by suitable choice of promoter sequences, or by selecting the number of copies or the site of integration of the DNA sequences that are introduced into the plant genome.
  • the DNA construct may include more than one SAM-decarboxylase DNA sequence or more than one recombinant construct may be transformed into each plant cell.
  • SAM-decarboxylase activity while also modifying the activity of one or more other enzymes.
  • the other enzymes may be involved in cell metabolism or in fruit development and ripening.
  • Cell wall metabolising enzymes that may be modified in combination with SAM-decarboxylase include but are not limited to: pectin esterase, polygalacturonase, ⁇ -glucanase.
  • Other enzymes involved in fruit development and ripening that may be modified in combination with SAM-decarboxylase include but are not limited to: ethylene biosynthetic enzymes, carotenoid biosynthetic enzymes, carbohydrate metabolism enzymes including invertase.
  • a first plant may be individually transformed with a SAM-decarboxylase construct and then crossed with a second plant which has been individually transformed with a construct encoding another enzyme.
  • plants may be either consecutively or co- transformed with SAM-decarboxylase constructs and with appropriate constructs for modification of the activity of the other enzyme(s) .
  • An alternative example is plant transformation with a SAM-decarboxylase construct which itself contains an additional gene for modification of the activity of the other enzyme(s).
  • the SAM-decarboxylase constructs may contain sequences of DNA for regulation of the expression of the other enzyme(s) located adjacent to the SAM-decarboxylase sequences. These additional sequences may be in either sense or antisense orientation as described in International patent application publication number W093/23551 (single construct having distinct DNA regions homologous to different target genes) . By using such methods, the benefits of modifying the SAM-decarboxylase activity may be combined with the benefits of modifying the activity of other enzymes.
  • a method for modifying SAM-decarboxylase gene expression in plants by transforming plants with SAM-decarboxylase DNA constructs and growing such transformed plants or their descendants followed by selection of plants having modified SAM-decarboxylase gene expression.
  • Suitable SAM-decarboxylase DNA constructs may be adapted to enhance the production of the SAM-decarboxylase enzyme or to inhibit such production by the plant when compared with untransformed plants.
  • This method may be used for modifying senescence characteristics: plants are transformed with SAM-decarboxylase DNA constructs, the transformed plants or their descendants are grown and plants having modified senescence characteristics are selected.
  • This method may also be used for modifying fruit-ripening characteristics: fruit-bearing plants are transformed with SAM-decarboxylase DNA constructs, the transformed plants or their descendants are grown and plants having modified fruit-ripening characteristics are selected.
  • plants produced by the method of the invention may also contain other recombinant constructs, for example constructs having other effects on fruit ripening (such as constructs inhibiting the production of polygalacturonase or pectinesterase, or interfering with ethylene production) .
  • Fruit containing both types of recombinant construct may be made either by successive transformations, or by crossing two varieties that each contain one of the constructs and selecting among the progeny for those that contain both.
  • Figure 1 shows the ethylene and polyamine biosynthetic pathways
  • FIG. 2 is a diagram showing the construction Of pRNGUP16; and with reference to the SEQUENCE LISTING in which:
  • SEQ ID NO 1 shows the sequence of the GUP16 cDNA
  • SEQ ID NO 2 shows the sequence of the protein encoded by GUP16.
  • EXAMPLE 1 Construction of a tomato wild-type ripening fruit cDNA library.
  • Tomato plants (Lvcopersicon esculentum Mill, cv Ailsa Craig, +/+ genotype) were grown under standard conditions. Flowers were tagged at anthesis and fruit removed from the plant, at a very early-breaker stage, when changes in pigmentation of the fruit were first apparent. This stage corresponded to 43 to 48 days post-anthesis. Pericarp samples were frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted from the pooled pericarp of approximately four fruit as has been previously described. Poly(A) mRNA was isolated by oligo dT Cellulose chromatography using a POLY(A)QUIK mRNA purification kit according to the manufacturers protocol (Stratagene,CA,USA) .
  • Double stranded cDNA was synthesised from approximately 5 ⁇ g mRNA using a 1 ZAP cDNA synthesis kit.
  • cDNA was ligated into 1 UNI-ZAP II, encapsidated in vitro and amplified immediately in E coli strain XLl-Blue following the manufacturers instructions (Stratagene, CA, USA) .
  • the wild-type tomato fruit cDNA library was estimated to represent >10 primary recombinants. Following a single amplification step, non- recombinants were estimated at less than 3% of the total recombinants. Insert size of cloned cDNAs, obtained following PCR of randomly selected clones using T3 and T7 oligonucleotide primers, was estimated to be between 500bp and 2.2Kb with a median of approximately 1.0Kb. Approximately 1.8 x
  • the GUP16 clone was picked randomly and identified following plaque purification and subsequent in vivo excision of the cloned insert. Similar clones may be isolated using a probe comprising the sequence shown as SEQ ID NO 1.
  • the sequence of the GUP16 cDNA clone is shown as SEQ ID NO 1 and is 1445 base pairs in length.
  • the GUP16 cDNA clone contains the complete coding sequence for a SAM-decarboxylase enzyme.
  • SEQ ID NO 2 shows the amino acid sequence of the protein encoded by GUP16, as deduced from the DNA sequence. Computer searches with SEQ ID NO 2 indicate strong homology with animal SAM-decarboxylase enzymes.
  • the cDNA clone (TUB13) isolated from potato Solanum tuberosum is also homologous to a human SAM decarboxylase gene (Taylor et al, 1992, Plant Molecular Biology, 20:641-651; EMBL/GenBank accession number Z11680) and to unsequenced genes in the potato Solanum brevidens and in the tomato Lycopersicon esculentum.
  • GUP16 As a hybridisation probe, it has been shown that GUP16 mRNA is expressed in young tomato fruit and declines during later stages of fruit development and ripening.
  • EXAMPLE 4 Construction of antisense RNA vectors with the CaMV 35S promoter.
  • a vector is constructed using sequences corresponding to a restriction fragment obtained from GUP16 and is cloned into the vectors GA643 (An et al, 1988, Plant Molecular Biology Manual A3: 1-19) or pDH51 (Pietrzak et. al, 1986, Nucleic Acids Research, 14:5875-5869) which has previously been cut with a compatible restriction enzyme (s) .
  • a restriction fragment from the GUP16/pDH51 clone containing the promoter, the GUP16 fragment and other pDH51 sequence is cloned into SLJ44026B or SLJ44024B (Jones et. al, 1990, Transgenic Research, 1) or a Binl9 (Bevan, 1984, Nucleic Acids Research, 12:8711-8721) which permits the expression of the antisense RNA under control of the CaMV 35S promoter.
  • EXAMPLE 5 Construction of antisense RNA vectors with the polygalacturonase promoter.
  • pJR3 is a Binl9 based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase promoter.
  • This vector includes approximately 5 kb of promoter sequence and 1.8 kb of 3' sequence from the PG promoter separated by a multiple cloning site.
  • the fragment of GUP16 cDNA described in Example 4 is also cloned into the vectors described in Example 4 in the sense orientation. After synthesis, the vectors with the sense orientation of GUP16 sequence are identified by DNA sequence analysis.
  • EXAMPLE 7 Construction of truncated sense RNA vectors with the polygalacturonase promoter.
  • the fragment of GUP16 cDNA that was described in Example 4 is also cloned into the vector pJR3 in the sense orientation. After synthesis, the vectors with the sense orientation of GUP16 sequence are identified by DNA sequence analysis.
  • EXAMPLE 8 Construction of a GUP16 over-expression vector using the CaMV35S promoter.
  • the complete SAM decarboxylase cDNA sequence is inserted into the vectors described in Example 4 to give an over-expression vector.
  • EXAMPLE 9 Construction of a GUP16 over-expression vector using the polygalacturonase promoter.
  • FIG. 2 is a diagram showing the construction of a DNA vector, pRNGUP16.
  • GUP16 cDNA is removed by digestion with Smal and BamHI and the fragment made blunt with Klenow polymerase and ligated into pRN2 (containing the PG promoter) cut with BamHI and made blunt with klenow.
  • Vectors are transferred to Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and are used to transform tomato plants.
  • Transformation of tomato cotyledons follows standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988) . Transformed plants are identified by their ability to grow on media containing the antibiotic kanamycin. Plants are regenerated and grown to maturity.
  • Plant parts are analysed for modifications to their senescence characteristics.
  • Fruit are analysed for modifications to their ripening characteristics.

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Abstract

L'invention concerne un ADN homologue à une séquence codant une enzyme de carboxylase de SAM. Cet ADN est incorporé dans des produits de recombinaison d'ADN transformés en plantes, de façon à amplifier ou à réduire l'expression du gène correspondant. Ceci permet de réaliser un procédé de modification des caractéristiques de sénescence et de maturation de fruits.
EP95900873A 1993-11-18 1994-11-17 Adn, produits de recombinaison d'adn, cellules et plantes derivees desdits produits Withdrawn EP0729512A1 (fr)

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GB9323771 1993-11-18
GB939323771A GB9323771D0 (en) 1993-11-18 1993-11-18 Dna,dna consrtructs,cell and plants derived therefrom
PCT/GB1994/002532 WO1995014092A1 (fr) 1993-11-18 1994-11-17 Adn, produits de recombinaison d'adn, cellules et plantes derivees desdits produits

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GB9917875D0 (en) * 1999-07-30 1999-09-29 Du Pont Uk Nucleic acid constructs
AP2691A (en) 2004-08-18 2013-07-16 Alellyx Sa Polynucleotides, Dna constructs and methods for the alteration of plant lignin content and/or composition

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WO1995014092A1 (fr) 1995-05-26
GB9323771D0 (en) 1994-01-05
AU1032395A (en) 1995-06-06

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