EP0876481A1 - Genes associes au murissement des fruits - Google Patents

Genes associes au murissement des fruits

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Publication number
EP0876481A1
EP0876481A1 EP97900698A EP97900698A EP0876481A1 EP 0876481 A1 EP0876481 A1 EP 0876481A1 EP 97900698 A EP97900698 A EP 97900698A EP 97900698 A EP97900698 A EP 97900698A EP 0876481 A1 EP0876481 A1 EP 0876481A1
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EP
European Patent Office
Prior art keywords
sequence
strawberry
ripening
gene
protein
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.)
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EP97900698A
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German (de)
English (en)
Inventor
Kenneth Manning
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Horticulture Research International
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Horticulture Research International
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Filing date
Publication date
Priority claimed from GBGB9601330.5A external-priority patent/GB9601330D0/en
Priority claimed from GBGB9618742.2A external-priority patent/GB9618742D0/en
Application filed by Horticulture Research International filed Critical Horticulture Research International
Priority to EP02028930A priority Critical patent/EP1321525A3/fr
Publication of EP0876481A1 publication Critical patent/EP0876481A1/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Withdrawn legal-status Critical Current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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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)
    • C12N15/8241—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 generally to the modification of a plant phenotype by the regulation of plant gene expression More specifically it relates to the control of fruit ripening by control of one or more than one gene which is known to be implicated in that process
  • the antisense fragment does not have to be precisely the same as the endogenous complementary strand of the target gene There simply has to be sufficient sequence similarity to achieve inhibition of the target gene
  • sequences isolated from one species may be effective in another, it is not infrequent to find exceptions where the degree of sequence similarity between one species and the other is insufficient for the effect to be obtained. In such cases, it may be necessary to isolate the species-specific homologue.
  • Antisense downregulation technology is well-established in the art It is the subject of several textbooks and many hundreds of journal publications The principal patent reference is European Patent No. 240,208 in the name of Calgene Ine There is no reason to doubt the operability of antisense technology It is well-established, used routinely in laboratories around the world and products in which it is used are on the market Both overexpression and downregulation are achieved by "sense" technology If a full length copy of the target gene is inserted into the genome then a range of phenotypes is obtained, some overexpressing the target gene, some underexpressing A population of plants produced by this method may then be screened and individual phenotypes isolated As with antisense, the inserted sequence is lacking in a translation initiation signal.
  • Sense and antisense gene regulation is reviewed by Bird and Ray in Biotechnology and Genetic Engineering Reviews 9 : 207-227 ( 1991 ). The use of these techniques to control selected genes in tomato has been described by Gray et al., Plant Molecular Biology, J9 69-87 (1992).
  • Agrobacterium- mediated transformation For dicotyledonous plants the most widely used method is Agrobacterium- mediated transformation. This is the best known, most widely studied and, therefore, best understood of all transformation methods
  • the rhizobacterium Agrobacterium tumefaciens, or the related Agrob cterium rhizogenes contain certain plasmids which, in nature, cause the formation of disease symptoms, crown gall or hairy root tumours, in plants which are infected by the bacterium.
  • Part of the mechanism employed by Agrobacterium in pathogenesis is that a section of plasmid DNA which is bounded by right and left border regions is transferred stably into the genome of the infected plant.
  • the effectiveness oi Agrobacterium is restricted to the host range of the micro ⁇ organism and is thus restricted more or less to dicotyledonous plant species.
  • monocotyledonous species which include the important cereal crops, are not amenable to transformation by the Agrobacterium method.
  • Various methods for the direct insertion of DNA into the nucleus of monocotyledon cells are known.
  • microparticles of dense material are fired at high velocity at the target cells where they penetrate the cells, opening an aperture in the cell wall through which DNA may enter.
  • the DNA may be coated on to the microparticles or may be added to the culture medium.
  • the DNA is inserted by injection into individual cells via an ultrafine hollow needle.
  • Another method applicable to both monocotyledons and dicotyledons, involves creating a suspension of the target cells in a liquid, adding microscopic needle-like material, such as silicon carbide or silicon nitride " whiskers", and agitating so that the cells and whiskers collide and DNA present in the liquid enters the cell
  • microscopic needle-like material such as silicon carbide or silicon nitride " whiskers”
  • whiskers agitating so that the cells and whiskers collide and DNA present in the liquid enters the cell
  • the requirements for both sense and antisense technology are known and the methods by which the required sequences may be introduced are known. What remains, then is to identify genes whose regulation will be expected to have a desired effect, isolate them or isolate a fragment of sufficiently effective length, construct a chimeric gene in which the effective fragment is inserted between promoter and termination signals, and insert the construct into cells ofthe target plant species by transformation. Whole plants may then be regenerated from the transformed cells.
  • This invention is concerned with the control of ripening in fruit, and the particular interest here is in strawberries.
  • the interest in controlling the ripening process is to improve the flavour and/or texture of the fruit, both characters being largely affected by the ripening process.
  • Sugars are the most important soluble component of the flavour. Some 99% of the soluble sugars in strawberry are accounted for by sucrose, glucose and fructose, the amount of these sugars being affected by the season but their relative proportions are largely unaffected.
  • An object of the present invention is to provide DNA sequences enabling the construction of vectors suitable for genetic transformation of strawberry plants, with a view to control of the ripening process in strawberry fruit.
  • a vector for use in the genetic transformation of strawberry cells comprising a promoter sequence, a regulation sequence and a transcription termination sequence, in which the regulation sequence comprises the coding region, or a fragment of at least 10 bases thereof, of a strawberry protein selected from O-methyl transferase, acyl carrier protein (ACP), elongation factor, auxin-induced gene, cysteine(thiol) proteinase, cellulase, starch phosphorylase, pyruvate decarboxylase, chalcone reductase, protein kinase, auxin- related gene, sucrose transporter, meristem pattern gene, or selected from a strawberry protein with homology to transcribed sequence accession number T45086, transcribed sequence accession number L36159 or transcribed sequence accession number T45902, or selected from a strawberry protein of unknown homology encoded by one of the StrawRipe sequences A to K.
  • ACP acyl carrier protein
  • elongation factor auxin-induced gene
  • the gene regulation sequence may be in the same or antisense orientation as the endogenous target gene. It may also be of partial or full sequence length.
  • the invention further contemplates the overexpression of one or more of the genes by inserting into the strawberry genome one or more than one extra copy thereof.
  • the invention also provides a gene regulation sequence which comprises the coding region, or a fragment of at least 10 bases thereof, of a strawberry protein selected from O-methyl transferase, acyl carrier protein (ACP), elongation factor, auxin-induced gene, cysteine(thiol) proteinase, cellulase, starch phosphorylase, pyruvate decarboxylase, chalcone reductase, protein kinase, auxin-related gene, sucrose transporter, meristem pattern gene, or selected from a strawberry protein with homology to transcribed sequence accession number T45086, transcribed sequence accession number L36159 or transcribed sequence accession number T45902, or selected from a strawberry protein of unknown homology encoded by one of the StrawRipe sequences A to K
  • sequences of this invention can also be used as probes for isolation of similar sequences from the strawberry genome
  • the invention also provides a strawberry plant and propagating material thereof which contains a vector of this invention.
  • a method for altering the phenotype of strawberry plants comprising inserting into the genome ofthe cell of a strawberry plant a gene regulation vector of this invention
  • the invention further provides genetically modified strawberry plants, propagation material and strawberry fruit
  • the regulation sequence comprises the coding region, or a fragment of at least 10 bases thereof, of a strawberry protein
  • the strawberry protein is selected from O-methyl transferase, acyl carrier protein (ACP), elongation factor, auxin-induced gene, cysteine(thiol) proteinase, cellulase, starch phosphorylase, pyruvate decarboxylase, chalcone reductase, protein kinase, auxin-related gene, sucrose transpo ⁇ er, meristem pattern gene, or selected from a strawberry protein with homology to transcribed sequence accession number T45086, transcribed sequence accession number L36159 or transcribed sequence accession number T45902, or selected from a strawberry protein of unknown homology encoded by one of the StrawRipe sequences A to K
  • SEQ LD NO 1 to SEQ LD NO.27 also referred to herein as Sequences 1 to 27 Related sequences taken from the priority documents of the present PCT application are given in SEQ LD NO 28 to SEQ LD NO 38
  • the gene regulation sequences of the invention may be synthesised from the sequence information given or may be isolated from a library To assist isolation Zeneca Limited have deposited with the National Collection of Industrial & Marine Bacteria, St Machar Drive, Aberdeen, UK, a cDNA library of strawberry ripening genes The library was deposited on 15th November 1994 under the Budapest Treaty and has the Accession Number NCIMB 40690
  • this invention is based on the identification of genes which encode proteins implicated in strawberry ripening-related processes DNA sequences which encode these proteins have been cloned and some have been characterised The DNA sequences may be used to modify plants with the goal of modifying the ripening characteristics of fruit.
  • strawberry plants can be generated which, amongst other phenotypic modifications, may have one or more of the following fruit characteristics
  • modified colour due to changes in activity of enzymes involved in the pathways of pigment biosynthesis (e.g lycopene, ⁇ -carotene, chalcones and anthocyanins), increased resistance to post-harvest pathogens such as fungi
  • enzymes involved in the pathways of pigment biosynthesis e.g lycopene, ⁇ -carotene, chalcones and anthocyanins
  • increased resistance to post-harvest pathogens such as fungi
  • the activity of the ripening-related proteins may be either increased or reduced depending on the characteristics desired for the modified plant part (fruit, leaf, flower, etc)
  • the levels of protein may be increased; for example, by inco ⁇ oration of additional genes
  • the additional genes may be designed to give either the same or different spatial and temporal patterns of expression in the fruit "Antisense” or “partial sense” or other techniques may be used to reduce the expression of ripening- related protein
  • each ripening-related protein or enzyme may be modified either individually or in combination with modification ofthe activity of one or more other ripening-related proteins/enzymes
  • the activities of the ⁇ pening-related proteins/enzymes may be modified in combination with modification of the activity of other enzymes involved in fruit ripening or related processes
  • 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 ripening-related sequences is described above; it is convenient to use DNA sequences derived from the ripening-related clones deposited at NCLMB in Aberdeen. Sequences coding for the whole, or substantially the whole, ofthe appropriate ripening-related protein 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 base sequence for transcription it is possible to use either intron or exon regions or a combination of both.
  • the regulation sequence varies from Sequences 1 to 27 but retains sufficient similarity to be effective in gene regulation.
  • the regulatory gene may be a homologue of a gene of Sequence 1 to 27 which has been obtained from a strawberry plant.
  • the cDNA sequence as found in one of the strawberry plasmids or the gene sequence as found in the chromosome ofthe strawberry 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.
  • Promoters suitable for use in constructs of the invention may be any suitable promoters which are known to be effective in driving expression of foreign genes in plants, for example the promoters may be those which are isolatable from the genomic version of the cDNAs of the invention.
  • 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 ripening-related RNA.
  • 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 ofthe original plant gene. Constructs expressing sense RNA encode RNA with a base sequence which is homologous to part or all of the sequence ofthe mRNA. In constructs which express the functional ripening-related protein, the whole ofthe coding region ofthe 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 is treated with 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, the polyubiquitin promoter and the tomato polygalacturonase gene promoter sequence (Bird et al, 1988, Plant Molecular Biology, 1 1 :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 ripening-related protein activity only during fruit development and/or ripening.
  • a constitutive promoter will tend to affect ripening-related protein levels and functions in all pans of the plant, while use of a tissue specific promoter allows more selective control of gene expression and affected functions.
  • tissue specific promoter allows more selective control of gene expression and affected functions.
  • the antisense or sense RNA is produced only in the organ in which its action is required and/or only at the time required.
  • Fruit development and/or ripening-specific promoters that could be used include the ripening-enhanced polygacturonase promoter (PCT WO 92/08798), the E8 promoter (Diekman & Fischer, 1988, EMBO, 7:3315-3320), the fruit specific 2AII promoter (Pear et al, 1989, Plant Molecular Biology, 13:639-651), the histidine decarboxylase promoter (HDC, Sibia) and the phytoene synthase promoter.
  • PCT WO 92/08798 the ripening-enhanced polygacturonase promoter
  • E8 promoter Diekman & Fischer, 1988, EMBO, 7:3315-3320
  • the fruit specific 2AII promoter Pear et al, 1989, Plant Molecular Biology, 13:639-6
  • Ripening-related protein or enzyme activity may be modified to a greater or lesser extent by controlling the degree of the appropriate ripening-related protein's 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 the ripening-related protein or more than one recombinant construct may be transformed into each plant cell.
  • each ripening-related protein may be separately modified by transformation with a suitable DNA construct comprising a ripening-related sequence
  • the activity of two or more ripening-related proteins may be simultaneously modified by transforming a cell with two or more separate constructs.
  • a plant cell may be transformed with a single DNA construct comprising both a first ripening-related sequence and a second ripening-related sequence.
  • ripening-related protein(s) it is also possible to modify the activity ofthe ripening-related protein(s) 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 a ripening-related protein include but are not limited to: pectin esterase, polygalacturonase, ⁇ -gal actanase, , ⁇ -glucanase.
  • enzymes involved in fruit development and ripening that may be modified in combination with a ripening-related protein include but are not limited to: ethylene biosynthetic enzymes, carotenoid biosynthetic enzymes including phytoene synthase, carbohydrate metabolism enzymes including invertase.
  • a first plant may be individually transformed with a ripening-related gene 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 ripening-related constructs and with appropriate constructs for modification ofthe activity ofthe other enzyme(s).
  • An alternative example is plant transformation with a ripening-related construct which itself contains an additional gene for modification of the activity of the other enzyme(s).
  • the ripening-related gene constructs may contain sequences of DNA for regulation of the expression of the other enzyme(s) located adjacent to the ripening-related sequences.
  • a DNA construct ofthe 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 ripening-related 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 isoenzyme gene expression).
  • Transgenic plants and their progeny may be used in standard breeding programmes, resulting in improved plant lines having the desired characteristics
  • fruit-bearing plants expressing a ripening-related construct according to the invention may be inco ⁇ orated into a breeding programme to alter fruit-ripening characteristics and/or fruit quality
  • Such altered fruit may be easily derived from elite lines which already possess a range of advantageous traits after a substantial breeding programme: these elite lines may be further improved by modifying the expression of a single targeted ripening-related protein enzyme to give the fruit a specific desired property.
  • fruit may be obtained by growing and cropping using conventional methods Seeds may be obtained from such fruit by conventional methods (for example, tomato seeds are separated from the pulp of the ripe fruit and dried, following which they may be stored for one or more seasons) Fertile seed derived from the genetically modified fruit may be grown to produce further similar modified plants and fruit.
  • the fruit derived from genetically modified plants and their progeny may be sold for immediate consumption, raw or cooked, or processed by canning or conversion to soup, sauce or paste. Equally, they may be used to provide seeds according to the invention.
  • the genetically modified plants may be heterozygous for the ripening-related DNA constructs.
  • the seeds obtained from self fertilisation of such plants are a population in which the DNA constructs behave like single Mendelian genes and are distributed according to Mendelian principles: e.g., where such a plant contains only one copy ofthe construct, 25% of the seeds contain two copies ofthe construct, 50% contain one copy and 25% contain no copy at all.
  • Mendelian principles e.g., where such a plant contains only one copy ofthe construct, 25% of the seeds contain two copies ofthe construct, 50% contain one copy and 25% contain no copy at all.
  • the offspring of selfed plants produce fruit and seeds according to the present invention, and those which do may themselves be either heterozygous or homozygous for the defining trait. It is convenient to maintain a stock of seed which is homozygous for the ripening-related DNA construct.
  • All crosses of such seed stock will contain at least one copy of the construct, and self-fertilized progeny will contain two copies, i.e. be homozygous in respect ofthe character.
  • Such homozygous seed stock may be conventionally used as one parent in Fl crosses to produce heterozygous seed for marketing.
  • Such seed, and fruit derived from it form further aspects of our invention.
  • a process of producing Fl hybrid seed comprises producing a plant capable of bearing genetically modified fruit homozygous for a ripening-related DNA construct, crossing such a plant with a second homozygous variety, and recovering Fl hybrid seed. It is possible according to our invention to transform two or more plants with different ripening-related DNA constructs and to cross the progeny of the resulting lines, so as to obtain seed of plants which contain two or more constructs leading to reduced expression of two or more fruit-ripening-related proteins
  • Figure 1 is a diagrammatic map of plasmid pBINCEL
  • Double stranded cDNAs were cloned into the ⁇ gtlO vector using the BRL cloning system (8287SA. Bethseda Research Laboratories, Paisley, Renfrewshire, UK) essentially as follows Internal EcoRI sites of the cDNAs were methylated using EcoRI methylase The DNA termini were repaired with T4 DNA polymerase and phosphorylated EcoRI linkers ligated to the cDNA with T4 ligase Excess linkers were digested and removed by column chromatography on DEAE-Sephadex The purified double stranded cDNAs with EcoRI termini were ligated into ⁇ gtlO vector DNA digested with EcoRI and dephosphorylated Vector DNA was then packaged using an in vitro packaging extract (Promega Co ⁇ oration, Southampton, UK) Recombinant bacteriophage were mixed with plating bacteria (E. coli C600 hflA 150) as described in the BRL protocol to determine titre, for library screening
  • the unamplified cDNA library from ripe strawberry was differentially screened using cDNA from fruit receptacle tissue at the ripe and white stages of ripeness A proportion of the library was plated at low density and duplicate plaque lifts made on to Hybond N nylon filters (Amersham) according to the manufacturer's instructions One filter was hybridised to ripe cDNA from white fruit and the duplicate filter hybridised to ripe cDNA.
  • Hybridisations were at high stringency using digoxigenin as a non-radioactive label (Boehringer Mannheim, Lewes, Hampshire, UK) Plaques hybridising preferentially to ripe cDNA were picked and replated at low density for a second round of selection by differential screening Single plaques from the second screening were picked and numbered as ripening-enhanced clones 1.5 Characterisation of the ripe cDNA library and ripening-enhanced clones
  • the ripe cDNA library was prepared with an efficiency of 3.03x 106 plaque-forming units per microgram of cDN A.
  • the size of the cDNA inserts in this library ranged from approximately 0.24 to 6 kbp with a mean insert size of approximately 1 4 kbp.
  • Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, Cheshire, UK) with forward and reverse primers specific for the ⁇ gtlO vector. Improved sequence data were obtained for clones with multiple inserts and clones with single inserts that did not produce good sequence data by subcloning into the phagemid vector pBK-CMV (Stratagene) vector for sequencing. From the sequenced clones, the following twenty-seven ripening-related clones were sele ⁇ ed. Comparison of these sequences with sequences in the
  • RNA was extracted from strawberry fruit during normal development and analysed by Northern blotting using standard procedures. The level of messenger RNA corresponding to the expression of O-methyl transferase, cysteine proteinase, acyl carrier protein and auxin induced gene were monitored in the receptacle at various time points between pollination and the overripe stage, between Day 1 and Day 19, and then at the stages of Turning, Orange, Ripe and Overripe Messenger RNA for O-methyl transferase appeared at Day 19, through to Overripe and was highest at Orange and Ripe. The messenger RNA for cysteine proteinase was low up to day 19, and then increased between the Turning and Overripe stages. The messenger RNA for Acyl carrier protein was low up to Day 19, and increased for Turning, Orange and Ripe. The messenger RNA for Auxin induced gene appeared around Day 16, and was highest between the Turning and Overripe Stages
  • the data provide evidence that O-methyl transferase, cysteine proteinase, acyl carrier protein and auxin induced gene are involved in the ripening process in normal fruit development.
  • a vector is constructed using the sequences corresponding to a fragment of one of the sequences 1 to 38, more especially one of the sequences 1 to 27.
  • This fragment is synthesised by the polymerase chain reaction using synthetic primers. The ends of the fragment are made flush with T4 polymerase and it is cloned into a derivative of the pBINPLUS vector (van Engelen et ai, Transgenic Research 4, 288-290 (1995)) containing the cauliflower mosaic virus (CaMV) 35S promoter-nopaline synthase (nos) 3' terminator cassette inserted into the Hindlll/EcoRI site.
  • CaMV cauliflower mosaic virus
  • nos 35S promoter-nopaline synthase
  • the plasmid pB NCEL is obtained which is derived from pBINPLUS and which contains cellulase cDNA in either the sense or antisense orientation
  • a diagrammatic map of the plasmid pB NCEL is given in Figure 1
  • an antisense extended sequence comprising the cellulase of SEQ LD:6 with the addition of a polyA tail of 17 bases was inserted to give a pBLNCEL antisense cellulase vector
  • a vector is constructed using a restriction fragment obtained from a strawberrv ripening-related clone. The fragment is blunt ended with T4 polymerase and is cloned into a derivative of the pBINPLUS vector.
  • pJR3 is a Bin 19 based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase (PG) 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
  • vectors with the correct orientation ofthe ripening- related sequences are identified by DNA sequence analysis.
  • Alternative fruit enhanced promoters (E8, 2A11 or any strawberry promoter) are substituted for the polygalactonurase promoter in pJR3 or for the CaMV 35S promoter in the modified pBINPLUS vector described in Example 2 to give alternative patterns of expression.
  • the fragment of the ripening-related cDNA that was described in Example 2 is also cloned into the vectors described in Example 2 in the sense orientation
  • the vectors with the sense orientation of the phytoene synthase sequence are identified by DNA sequence analysis.
  • the fragment of the ripening-related cDNA that was described in Example 3 is, also cloned into the vectors described in Example 3 in the sense orientation.
  • the vectors with the sense orientation of the ripening-related sequence are identified by DNA sequence analysis.
  • Vectors are transferred to Agrobacterium tumefaciens EHA105 (a kanamycin sensitive strain of an organism widely available to plant biotechnologists; Hood et al., Transgenic Research 2, 208-218 (1990)) and are used to transform strawberry plants.
  • Strawberry explants infected with Agrobacterium are grown on regeneration medium normally containing 100 mg/l kanamycin After three weeks, the explants are transferred to regeneration medium without kanamycin.
  • putatively transformed shoots are cultured on propagation medium for two weeks and then transformants are selected on medium containing 25 mg/l kanamycin. Regenerated plants containing the transgene are selected and grown to maturity.
  • Ripening fruit are analyzed for modifications to their ripening characteristics
  • transformed plants were produced in this way using the pBINCEL antisense cellulase fragment of Example 2
  • the presence of the transgene in the putative strawberry transformant was verified by PCR using genomic DNA from the transformant as template and primers from the 35S promoter and from the cellulase strand
  • the PCR products were separated by agarose gel electrophoresis and a fragment of -1400 base pairs was obtained that was identical in size to the PCR product obtained using the pBINCEL antisense cellulase vector DNA as template
  • the following sequences have been edited to remove vector bases and polyA regions, as appropriate.
  • GGAGAAGGAC CAAATGCCGA TGGACTTCAA GGGTGTGTGG GCAGACATGG 450
  • GCCAAGTAAC CAAGTTTAAG GTGGGGGATG AAGTGTATGG GGATCTCAAT 350
  • TTTTCAGTCC TAGAGACCGG AAAGTACCCN AACGGAGCAC GGNCTAATAG 350
  • CAAATGAGCA CATCTNTNGN AGGGGCCNTG GGGCTCCCCC TTTTGGNAAA 300
  • GTATATNGCA CGTCTTCTTC TTCTTCTTCT TCTTCTTCTT (ZTTTTGGT 100
  • TTGTTATTCT TCATCTTCTA CCCTAATATA CTCTTTGATA CATAAAAGTC 200
  • CAGCACTTTT CAAACAATAG
  • CAACTCAGTA GTCTTTACCC TCAGTAGTGA 250
  • GCTTTTTCTC CNTCAANGCN AATTCCCGTT NGNTNTTCTT NTTNTGCCNA 450
  • CNAGCCCATG TATGAANTGC TATTTGAGCG GCTTAATACG CNTCCCGGAG 450
  • AAAGTACATT AAAANTATGG ATATGCCCTG TNCTGAAATA TGACTGAAAA 300
  • TTCTCTACCA AAAAGACTCA GAATTTGCTC TCCTTTGGCT ACTATTCCTC 550 CGTCAGTCAA TCAANTTTAA NATGANTCCA TTTTGGCAAC AGAAGAACCT 600
  • TCAAACTTTA CATGTNCCAC ATTTTCAGAG GGCTGGCTTA CATACACACC 350

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Abstract

Vecteur à utiliser dans la transformation génétique de cellules de fraises, qui comprend une séquence promotrice, une séquence de régulation et une séquence de fin de transcription . Ladite séquence de régulation comprend la région codante, ou un fragment d'au moins 10 bases de ladite région, d'une protéine de fraise choisie parmi la O-méthyle transférase, la protéine transporteuse d'acyles, le facteur d'élongation, le gène induit par l'auxine, la cystéine(thiol) protéinase, la cellulase, la phosphorylase d'amidon, la pyruvate décarboxylase, la chalcone réductase, la protéine kinase, le gène associé à l'auxine, le transporteur du saccharose, le gène de configuration de méristème, ou d'une protéine de fraise présentant une homologie au numéro d'accès de séquence transcrite T45086, au numéro d'accès de séquence transcrite L36159 ou au numéro d'accès de séquence transcrite T45902, ou d'une protéine de fraise d'homologie inconnue codée par l'une des séquences du gène de mûrissement de la fraise (StrawRipe) A à K.
EP97900698A 1996-01-23 1997-01-21 Genes associes au murissement des fruits Withdrawn EP0876481A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02028930A EP1321525A3 (fr) 1996-01-23 1997-01-21 Genes pour la maturation des fruites

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GBGB9601330.5A GB9601330D0 (en) 1996-01-23 1996-01-23 Fruit ripening-related genes
GB9601330 1996-01-23
GBGB9618742.2A GB9618742D0 (en) 1996-09-09 1996-09-09 Fruit ripening-related genes
GB9618742 1996-09-09
PCT/GB1997/000178 WO1997027295A1 (fr) 1996-01-23 1997-01-21 Genes associes au murissement des fruits

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Publication number Priority date Publication date Assignee Title
US6080914A (en) * 1997-01-21 2000-06-27 Monsanto Company Strawberry promoters and genes
US6043410A (en) * 1998-02-06 2000-03-28 Calgene Llc Strawberry fruit promoters for gene expression
WO1999043833A1 (fr) * 1998-02-26 1999-09-02 Universität Bern Plantes transgeniques resistantes aux maladies
MXPA01000174A (es) * 1998-06-26 2003-09-10 Univ Iowa State Res Found Inc Materiales y metodos para la alteracion de los niveles de enzimas y de acetil-coa en plantas.
US6242668B1 (en) * 1998-08-06 2001-06-05 Dna Plant Technology Corporation Strawberry endo-1,4-β-glucanase genes and their uses
AU769932B2 (en) * 1998-12-02 2004-02-12 Plant Research International B.V. Fruit flavour related genes and use thereof
EP1006190A1 (fr) * 1998-12-02 2000-06-07 Centrum Voor Plantenveredelings- En Reproduktieonderzoek Gènes associés à l'arome des fruits et leurs applications
IL128111A (en) 1999-01-18 2007-03-08 Yissum Res Dev Co A method of silencing expression of a target sequence in a plant genome
FR2803600B1 (fr) * 2000-01-10 2002-04-05 Agronomique Inst Nat Rech Promoteur de plante fruit specifique
ES2164590B1 (es) * 2000-03-10 2005-01-01 Universidad De Cordoba Molecula de adn que codifica para una poligalacturonasa de fresa y susaplicaciones.
US7323621B2 (en) 2002-12-13 2008-01-29 E.I. Du Pont De Nemours And Company Method of decreasing liquiritigenin-derived isoflavones relative to total isoflavones in plants and plants producing reduced ratio of liquiritigenin-derived isoflavones relative to total isoflavones
WO2013148257A1 (fr) * 2012-03-30 2013-10-03 J.R. Simplot Company Formation de stéviol et de glycosides de stéviol dans des plantes
CN108977462B (zh) * 2018-08-24 2022-05-17 安徽省农业科学院园艺研究所 一种提高草莓果实中含糖量的方法
CN113999828B (zh) * 2021-02-02 2024-01-30 中国科学院植物研究所 草莓甲基转移酶mta和mtb基因在控制草莓成熟中的应用
CN114561400A (zh) * 2022-03-07 2022-05-31 安徽农业大学 红颜草莓硝酸盐转运蛋白基因FaNRT1.1及其应用

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GB9009307D0 (en) * 1990-04-25 1990-06-20 Ici Plc Dna,constructs,cells and plant derived therefrom
GB9018612D0 (en) * 1990-08-24 1990-10-10 Ici Plc Dna,constructs,cells and plants derived therefrom
EP0564524A1 (fr) * 1990-12-26 1993-10-13 Monsanto Company Regulation du murissement des fruits et de la senescence chez les plantes
GB9320930D0 (en) * 1993-10-12 1993-12-01 Zeneca Ltd Modified fruit

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Title
See references of WO9727295A1 *

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WO1997027295A1 (fr) 1997-07-31
AU1316297A (en) 1997-08-20
EP1321525A3 (fr) 2003-09-10

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