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WO1994028180A2 - Fruit dont l'activite de l'enzyme malique liee au nadp est modifiee - Google Patents

Fruit dont l'activite de l'enzyme malique liee au nadp est modifiee Download PDF

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Publication number
WO1994028180A2
WO1994028180A2 PCT/GB1994/001197 GB9401197W WO9428180A2 WO 1994028180 A2 WO1994028180 A2 WO 1994028180A2 GB 9401197 W GB9401197 W GB 9401197W WO 9428180 A2 WO9428180 A2 WO 9428180A2
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Prior art keywords
nadp
fruit
sequence
dna
plant
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PCT/GB1994/001197
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English (en)
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WO1994028180A3 (fr
Inventor
Colin Roger Bird
Graham Barron Seymour
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Zeneca Limited
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Priority to AU68042/94A priority Critical patent/AU6804294A/en
Publication of WO1994028180A2 publication Critical patent/WO1994028180A2/fr
Publication of WO1994028180A3 publication Critical patent/WO1994028180A3/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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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

Definitions

  • This invention relates to the modification of malate metabolism in fruit using DNA constructs.
  • 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 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 into the genome of the plant of a synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of individual genes involved in the development and ripening of tomato fruit (Gray et al, 1992, Plant Molecular Biology, 19:69-87).
  • 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.
  • Malic acid (C0 2 H.CHOH.CHOH.C0 2 H) is an important constituent of tomato fruit which is involved in several metabolic pathways that influence ripe fruit quality. During ripening, the levels of malic acid decline. However, it has been proposed that the products of its metabolism play a major role in the ripening of fruit.
  • malate CC- 2 ⁇ .CHOH.CHOH.C0 2 "
  • malate may serve as a source of carbon and reductive power for gluconeogenesis (Halinska and Frenkel, 1990, Plant Physiol. 95, 954-960).
  • NADP- ME NADP-linked malic enzyme
  • EC 1.1.1.40 The decarboxylation of malate catalysed by NADP-linked malic enzyme
  • tomato fruit the respiratory climacteric and the onset of ripening are accompanied by a decrease in the levels of malate in the fruit.
  • a large rise in the synthesis of NADP-linked malic enzyme was reported to accompany these changes (Goodenough et al, 1985, Phytochem. 24, 1157-1162; Goodenough, 1990, Goodenough, 1990, In: The Flavour of Fruits, EDs I.D. Morton and A.J.Macleod) .
  • NADP-linked malic enzyme activity may be more closely associated with growth and accumulation of solutes rather than ripening (Knee and Finger, 1992, J. Amer. Soc. Hort. Sci. 117, 799-801).
  • NADP-linked malic enzyme is known to be present in various plant species where it has a role in the decarboxylation of malate in various tissues.
  • Genes encoding NADP-linked malic enzyme have been isolated from maize, bean, poplar and Flavia trinervia (Rothermel and Nelson, 1989, J Biol Chem, 264: 19587-19592; Walter et al, 1990, Plant Mol Biol, 15:525-526; Doorsselaere et al, 1991, Plant Physiol, 96:1385-1386; Borsch and Westhoff, 1990, FEBS Lett, 273:111-115). These NADP-linked malic enzyme clones have been sequenced but none of them are derived from fruit.
  • a method for producing plants having modified fruit characteristics which comprises transformation of fruit-bearing plants with a DNA construct adapted to modify the activity of NADP- linked malic enzyme and subsequent selection of plants having modified NADP-linked malic enzyme activity.
  • the characteristics of the ripening and/or mature fruit may be modified.
  • the levels of NADP- linked malic enzyme may be either reduced or increased during fruit development and ripening depending on the characteristics desired for the modified fruit.
  • “Antisense” or “partial sense” or other techniques may be used to reduce the expression of NADP-linked malic enzyme in developing and ripening fruit.
  • the level of NADP- linked malic enzyme (NADP-ME) may also be increased; for example, by incorporation of additional NADP-ME genes.
  • the additional genes may be designed to give either the same or different spatial and temporal patterns of expression in the fruit.
  • Any source of NADP-linked malic enzyme gene may be used as suitable genes may be isolated from any plant species or from bacteria, yeast, lower and higher eukaryotes. However, use of a fruit-derived NADP-linked malic enzyme sequence in the DNA constructs may allow more effective control of the endogenous genes expressed in the fruit, thus allowing more effective modification of fruit characteristics.
  • Reduced activity of NADP-ME can be used to modify various aspects of fruit quality, including:
  • Increased activity of NADP-ME can be used to modify various aspects of fruit quality, including: (a) Modified flavour due to modified levels of organic acid and changes in the sugar acid/balance in the fruit.
  • NADP-ME NADP-linked malic enzyme
  • NADP-linked malic enzyme activity may lead to modified levels of malic acid content in the fruit.
  • our previous International Patent Application Publication Number W092/17596 describes the inhibition of the TOM75 gene using an antisense construct to give tomato fruit of greater acidity and hence altered taste (transgenic fruit have a 25% greater malic acid content).
  • the TOM75 gene is not related to TMAL: its exact identity is unknown but it is likely to encode an integral membrane protein involved in metabolite transport.
  • TMAL encodes NADP-linked malic enzyme, which has a specific role and is active during fruit ripening. Modifying the expression of NADP-linked malic enzyme (for example, using TMAL constructs) is therefore a more direct and predictable method to modify fruit characteristics.
  • the invention further provides a DNA construct comprising a DNA sequence corresponding to at least part of a NADP-linked malic enzyme gene obtainable from fruit, the DNA sequence being preceded by a transcriptional initiation region operative in plants so that the construct can generate RNA in plant cells.
  • TMAL cDNA clone was deposited on 27 May 1993 1993 under the terms of the Budapest Treaty at the National Collections of Industrial and Marine Bacteria (23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland) and was given the reference NCIMB 40562.
  • a cDNA clone similar to pTMAL may alternatively be obtained from the mRNA of tomatoes or other fruit by a method similar to that described by Slater e_t al (1985, Plant Molecular Biology, 5:137-147). Sequences coding for the whole, or substantially the whole, of the mRNA produced by the NADP-ME gene may thus be obtained. Suitable lengths of the cDNA so obtained may be cut out for use by means of restriction enzymes.
  • SEQ ID NO 1 shows the sequence of the 1Kb fragment (1010 bp) of pTMAL (the tomato fruit NADP- linked malic enzyme cDNA clone). From base number 1 to 1010 there is a continuous open reading frame encoding the partial sequence of NADP-linked malic enzyme.
  • pTMAL shows homology (approximately 77-86%) to NADP-ME genes from maize, bean, poplar and Flavia trinervia (Rothermel and Nelson, 1989, J Biol Chem, 264: 19587-19592; Walter et al, 1990, Plant Mol Biol, 15:525-526; Doorsselaere et al, 1991, Plant Physiol, 96:1385-1386; Borsch and Westhoff, 1990, FEBS Lett, 273:111-115).
  • An alternative source of DNA for the base sequence for transcription is a suitable gene encoding the NADP-linked malic enzyme.
  • This gene may differ from the NADP-ME 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 NADP-ME 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 NADP-ME enzyme or any other protein. These promoters may be particularly responsive to ripening events and conditions.
  • NADP-ME promoters may be used to drive expression of any target gene.
  • a further way of obtaining a NADP-ME DNA sequence is to synthe ⁇ ise it ab initio from the appropriate bases, for example using SEQ ID NO 1 as a guide.
  • the preferred DNA sequence for use in the method and constructs of the present invention is DNA derived from the clone pTMAL or the corresponding TMAL gene or a synthetic polynucleotide based on the TMAL sequence.
  • DNA derived from the clone pTMAL or the corresponding TMAL gene or a synthetic polynucleotide based on the TMAL sequence is DNA derived from the clone pTMAL or the corresponding TMAL gene or a synthetic polynucleotide based on the TMAL sequence.
  • other sources of NADP-linked malic enzyme genes may also be used as suitable genes may be isolated from the fruit of other plant species.
  • NADP-ME sequences are incorporated into sense or antisense DNA constructs suitable for plant transformation. These DNA constructs may then be used to modify NADP-ME gene expression in plants.
  • the activities of the NADP-ME enzyme may also be modified in combination with modification of the activity of other cell wall enzymes or enzymes involved in fruit ripening.
  • the characteristics of fruit 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 showing modified fruit characteristics; 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 NADP-ME 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 to over-express the enzyme.
  • the constructs of the invention may be inserted into plants to regulate the production of NADP-linked malic enzymes encoded by genes homologous to TMAL.
  • the constructs may be transformed into any dicotyledonous or monocotyledonous plant.
  • the production of the enzyme 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 NADP-ME mRNA.
  • Constructs containing an incomplete DNA sequence substantially 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.
  • 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 NADP-ME (making the DNA construct a full or partial antisense construct).
  • 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. 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. 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 NADP-ME-encoding sequences is described above. Sequences coding for the whole, or substantially the whole, of the appropriate enzyme may thus be obtained. Suitable lengths of this DNA sequences may be cut out for use by means of restriction enzymes.
  • the cDNA sequence as found in the 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 NADP-ME 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 NADP-ME 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 clone R3.1
  • 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 (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 NADP-ME activity only during fruit development and/or ripening. .
  • a constitutive promoter will tend to affect NADP-ME 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 (malate metabolism).
  • a tissue specific promoter allows more selective control of gene expression and affected functions (malate metabolism).
  • ripening-specific promoters that could be used include the ripening-enhanced polygacturonase 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).
  • Malate metabolism may be modified to a greater or lesser extent by controlling the degree of NADP-ME 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 DNA sequence encoding NADP-ME or more than one recombinant construct may be transformed into each plant cell.
  • NADP-ME enzyme it is also possible to modify the activity of the NADP-ME enzyme 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 NADP-ME include but are not limited to: pectin esterase, polygalacturonase, ⁇ -galactanase, (3-glucanase.
  • Enzymes involved in fruit development and ripening that may be modified in combination with NADP-ME include but are not limited to: ethylene biosynthetic enzymes, carotenoid biosynthetic enzymes including phytoene synthase, carbohydrate metabolism enzymes including invertase.
  • NADP-ME Several methods are available for modification of the activity of NADP-ME in combination with other enzymes. For example, a first plant may be individually transformed with a NADP-ME construct and then crossed with a second plant which has been individually transformed with a construct encoding another enzyme. As a further example, single plants may be either consecutively or co- transformed with NADP-ME constructs and with appropriate constructs for modification of the activity of the other enzyme(s).
  • An alternative example is plant transformation with a NADP-ME construct which itself contains an additional gene for modification of the activity of the other enzyme(s).
  • the NADP-ME constructs may contain sequences of DNA for regulation of the expression of the other enzyme(s) located adjacent to the NADP-ME enzyme sequences.
  • 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. Suitable plants include any fruit-bearing plant (such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas and melons).
  • the NADP-ME sequence used in the transformation construct may be derived from the same plant species, or may be derived from any other plant species (as there will be sufficient sequence similarity to allow modification of related enzyme gene expression).
  • 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 Agrobacterium 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.
  • a 1Kb fragment of a tomato NADP-ME cDNA was isolated from a ripe tomato fruit cDNA library by PCR.
  • the primers for the PCR reaction were designed from highly conserved regions of previously characterised NADP-ME amino acid sequences from maize, bean, poplar and Flavia trinervia (Rothermel and Nelson, 1989, J Biol Chem, 264: 19587-19592; Walter et al, 1990, Plant Mol Biol, 15:525-526; Doorsselaere et al, 1991, Plant Physiol, 96:1385-1386; Borsch and Westhoff, 1990, FEBS Lett, 273:111-115).
  • the PCR product was isolated from a gel and the ends of the fragment were made flush with Klenow polymerase.
  • the fragment was then cloned into the vector Bluescript M13+, which had previously been cut with Smal . This clone was then characterised by sequence analysis.
  • SEQ ID NO 1 shows the 1010 bp sequence
  • a vector is constructed using the sequences corresponding to a fragment of the insert of the NADP-ME cDNA (pTMAL) .
  • This fragment is synthesised by polymerase chain reaction using synthetic primers. The ends of the fragment are made flush with T4 polymerase and it is cloned into the vector pJRl which has previously been cut with Smal.
  • pJRl (Smith et al, 1988, Nature, 334:724-726) is a Binl9 (Bevan, 1984, Nucleic Acids Research, 12:8711-8721) based vector, which permits the expression of the antisense RNA under the control of the CaMV 35S promoter.
  • This vector includes a nopaline synthase (nos) 3' end termination sequence.
  • pJR3 is a Binl9 based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase (PG) promoter.
  • PG tomato polygalacturonase
  • vectors with the correct orientation of the NADP-ME sequences are identified by DNA sequence analysis.
  • Alternative fruit enhanced promoters (such as E8 or 2A11) are substituted for the polygalacturonase promoter in pJR3 to give alternative patterns of expression.
  • the fragment of the NADP-ME cDNA that was described in Example 2 is also cloned into the vector pJRl in the sense orientation.
  • the vectors with the sense orientation of the NADP-ME sequence are identified by DNA sequence analysis.
  • EXAMPLE 5 Construction of truncated sense RNA vectors with fruit-enhanced promoter.
  • the fragment of the NADP-ME cDNA that was described in Example 2 is also cloned into the vector pJR3 in the sense orientation.
  • the vectors with the sense orientation of the NADP-ME sequence are identified by DNA sequence analysis.
  • NADP-ME cDNA containing a full open-reading frame is inserted into pJR3 or alternatives with different promoters
  • 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 stem segments follow standard protocols (e.g. Bird et al, 1988, Plant Molecular Biology, 11:651-662). Transformed plants are identified by their ability to grow on media containing the antibiotic kanamycin. Plants are regenerated and grown to maturity. Ripening fruit are analysed for modifications to their ripening characteristics.
  • the first 600 base pairs of pTMAL were used to make the antisense construct pTMALB with the 2A11 fruit specific promoter (Pear et al, 1989, Plant Molecular Biology, 13:639-651) and nos terminator.
  • the 2A11 promoter expresses throughout fruit development as well as ripening.
  • pTMALB constructs were used to transform tomato (variety Ailsa Craig) plants. Thirty individual transformants were generated. Sixteen of these have already started to fruit. Fruit were tagged at anthesis and harvest 35 days later (fruit were still green at this stage).
  • NADP-ME activity has been determined for an individual fruit from each plant. Fruit from two plants showed reduced NADP-ME enzyme activity: Plant 1.2 has 45% of wild-type NADP-ME activity; Plant 2.2 has undetectable NADP-ME activity. Fruit from the other plants showed approximately wild type NADP-ME activity. SEQUENCE LISTING
  • CAACAGTTGG TGAGGCTTGC CAGAAGTATG GAGGCATTTT CAGGCGTCCT CAGGGTCTCT 300

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Abstract

On modifie des caractéristiques de fruits en transformant des plantes à fruits avec un produit d'assemblage d'ADN adapté de façon à modifier l'activité de l'enzyme malique liée au NADP. Ces produits d'assemblage de l'ADN, qui peuvent être sens ou antisens, codent une partie de l'enzyme au moins. On décrit aussi des produits d'assemblage d'ADN qui comprennent une séquence d'ADN correspondant à une partie au moins du gène de l'enzyme malique, liée au NADP, qu'on peut obtenir d'un fruit.
PCT/GB1994/001197 1993-06-02 1994-06-02 Fruit dont l'activite de l'enzyme malique liee au nadp est modifiee WO1994028180A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU68042/94A AU6804294A (en) 1993-06-02 1994-06-02 Fruit with modified nadp-linked malic enzyme activity

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GB939311346A GB9311346D0 (en) 1993-06-02 1993-06-02 Modified fruit
GB9311346.2 1993-06-02

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WO1994028180A2 true WO1994028180A2 (fr) 1994-12-08
WO1994028180A3 WO1994028180A3 (fr) 1995-02-02

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6653535B1 (en) * 1999-05-28 2003-11-25 Pioneer Hi-Bred International, Inc. Methods for modulating water-use efficiency or productivity in a plant by transforming with a DNA encoding a NAPD-malic enzyme operably linked to a guard cell or an epidermal cell promoter
CN119639776A (zh) * 2025-01-16 2025-03-18 沈阳农业大学 一种南果梨PuNADP-ME基因、过表达和沉默载体及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9106713D0 (en) * 1991-03-28 1991-05-15 Ici Plc Dna,dna constructs,cells and plants derived therefrom

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6653535B1 (en) * 1999-05-28 2003-11-25 Pioneer Hi-Bred International, Inc. Methods for modulating water-use efficiency or productivity in a plant by transforming with a DNA encoding a NAPD-malic enzyme operably linked to a guard cell or an epidermal cell promoter
CN119639776A (zh) * 2025-01-16 2025-03-18 沈阳农业大学 一种南果梨PuNADP-ME基因、过表达和沉默载体及其应用

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GB9311346D0 (en) 1993-07-21
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