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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8237—Externally regulated expression systems
  • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
• • 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)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/823—Reproductive tissue-specific promoters
  • C12N15/8235—Fruit-specific

Definitions

• the present invention relates to the expression of genes in transgenic plants.
• it is concerned with the isolation and use of DNA sequences which control the expression of foreign genes in ripening fruits and in response to ethylene.
• the pTOM5 gene is involved in the step or steps of the pathway between geranylgeranyl pyrophosphate and phytoene, and that the pTOMS gene product is the enzyme known as phytoene synthase.
• the products produced by this branch of the pathway are carotenes, lutein, xanthophylls, and pigments such as lycopene, as well as plant growth regulators such as IBA.
• IBA plant growth regulators
• phytoene synthase mRNA is expressed in ripening tomato fruit. No expression could be detected in green fruit.
• the phytoene synthase gene is expressed most strongly at the full orange stage of ripening. The level of mRNA then declines in line with the general decline in biosynthetic capacity of the ripening fruit. Expression of phytoene synthase mRNA could also be induced by exposing mature green fruit to exogenous ethylene.
• the expression of the phytoene synthase gene is reduced in the Ripening Inhibitor (rin) and Noneripe (Nr) tomato fruit ripening mutants, which mature very slowly and never achieve the full red colour of ordinary tomato fruit.
• the genomic locations in the tomato of sequences homologous to the pTOM5 clone have been identified using RFLP mapping: two loci, on chromosome 2 and chromosome 3 respectively, carry sequences homologous to the pTO 5 clone. It has also been shown by Southern blotting that the pTOM5 gene may be present as a small multigene family.
• the present invention proposes to use the promoters of the phytoene synthase and similar genes to control the expression of novel and exogenous proteins and genes in tomato fruit.
• a DNA construct for use in transforming plant cells which comprises an exogenous coding sequence under the control of upstream promoter and downstream terminator sequences, characterised in that the upstream promoter has homology to a promoter of a gene of the carotenoid biosynthesis pathway.
• upstream promoter has homology to a promoter of a gene of the carotenoid biosynthesis pathway.
• Promoters for use in the invention may be derived from genes such as phytoene desaturase, cyclase and epoxydase. Such promoters may be isolated from genomic libraries by the use of cDNA probes, as has been done in the case of pTOM5. We particularly prefer to use the promoter of the phytoene synthase gene.
• downstream (3') terminator sequences can also be derived from the phytoene synthase gene: or they can be derived from other genes such as the polygalacturonase gene (see UK Patent Application 9025323.9 filed 8 November 1990). Many other possibilities are available from the literature.
• exogenous coding sequence' we indicate a sequence of DNA, other than that which follows the promoter region in the natural pTOM5 gene, that is adapted to be transcribed into functional RNA under the action of plant cell enzymes such as RNA polymerase.
• Functional RNA is RNA which affects the biochemistry of the cell: it may for example be mRNA which is translated into protein by ribosomes; or antisense RNA which inhibits the translation of mRNA complementary (or otherwise related) to it into protein.
• all kinds of exogenous coding sequences are useful in the present invention.
• this protein may be of bacterial origin - _> -
• phytoene synthase gene promoter to respond to exogenously supplied ethylene.
• exogenous coding sequences A wide variety of exogenous coding sequences is known from the literature, and the present invention is applicable to these as well as many others yet to be reported.
• the exogenous gene may code for RNA that interferes with the function of any kind of mRNA produced by the plant cell: for example, antisense RNA complementary to mRNA for fruit ripening genes such as polygalacturonase, pectinesterase, 3-1,4-glucanase, pTOMl3 etc.
• antisense RNA complementary to mRNA for fruit ripening genes such as polygalacturonase, pectinesterase, 3-1,4-glucanase, pTOMl3 etc.
• promoter sequences upstream - i.e. 5' - of the coding sequence of the gene
• Plant cells according to the invention may be transformed with constructs of the invention according to a variety of known methods (Agrobacterium Ti plasmids, electroporation, microinjection, microprojectile gun, etc).
• the transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way, although the latter are usually more easy to regenerate.
• genetically modified plants according to the present invention include, as well as tomatoes, fruits such as mangoes, peaches, apples, pears, strawberries, bananas and melons; and field crops such as maize (corn), sunflowers, sugarbeet, canola, and smallgrain cereals such as wheat, barley and rice.
• tomatoes fruits such as mangoes, peaches, apples, pears, strawberries, bananas and melons
• field crops such as maize (corn), sunflowers, sugarbeet, canola, and smallgrain cereals such as wheat, barley and rice.
• Plants produced by the process of the invention may contain more than one recombinant construct.
• constructs containing the phytoene synthase promoter they may contain a wide variety of other recombinant constructs, for example constructs having different effects on fruit ripening.
• these may be of enhanced colour (as a result of inserting extra gene copies of the PS gene and thereby overexpressing phytoene synthase) and may also contain constructs inhibiting the production of enzymes such as polygalacturonase and pectinesterase, or interfering with ethylene production (eg from pTOMl3, see PCT Application 90/01072 filed 12 July 1990).
• Tomatoes containing more than one type of recombinant construct may be made either by successive transformations, or by successively crossing varieties that each contain one of the constructs, and selecting among the progeny for those that contain all the desired constructs.
• a further aspect of the present invention is a process of activating exogenous coding sequences in plants under the control of the phytoene synthase promoter which comprises the application of exogenous ethylene.
• This may find particular use when fruit is stored in the absence of ethylene, and ethylene is then used to switch on the production of a given useful character providing extra value to the fruit at the point of sale. This may lead tc increase in sweetness of the fruit, or the production of special flavours or aromas, or the production of special polypeptides desired by the consumer. This will enable more flexibility in control of the fruit ripening process, particularly at the point of sale.
• Genomic clones representing two individual genes have been isolated and characterised by DNA sequence analysis.
• the clone gTOM5 represents part of a gene with exon sequence identical to the clone pTOM5.
• Clone F contains a sequence similar but not identical to pTOM5. Details of these clones are given below. Sequence and expression data suggest that Clone F encodes an untranscribed pseudogene.
• the genomic clones described in the Examples cover most of the coding region and the complete transcriptional initiation region of the phytoene synthase gene.
• the clone gTOM5 has been deposited at the National Collections of Industrial and Marine Bacteria (NCIB), now at 23 St. Machar Drive, Aberdeen AB2 1RY,
• Figures 1 and 1A show the nucleotide sequence of the 3.5 kb EcoRI - Sail fragment of gTOM5 (SEQ ID: 1) and the 3' region of the phytoene synthase gene (SEQ ID: 2);
• Figure 2 is a diagram of the structure of the phytoene synthase gene
• Figure 3 outlines a scheme for polymerase chain reaction amplification of the phytoene synthase gene promoter fragment
• Figure 4 outlines a scheme for construction of the plant transformation vector p5TAK.
• a library was constructed from tomato ( ycopersicon esculentum var. Ailsa Craig) genomic DNA which was partially digested with Sau3A and cloned into lambda EMBL3 (Bird et al (1988) Plant Molecular Biology 11, 651-662).
• the library was screened with the pTOM5 cDNA insert (Ray et al (1987) Nucleic Acids Research 1_5, 10587) and positive phages were purified by four successive cycles of plaque purification. Five positive clones were isolated. Restriction fragment mapping and DNA sequence analysis of these clones indicated that all 5 clones were overlapping and related. The clones did not have 100% sequence homology to pTOM5 in the regions that probably represented exons. This indicated that these clones represented a gene (designated clone F) that was not the pTOM ⁇ gene.
• oligonucleotides were designed that hybridised specifically to either pTOM5 or the clone F.
• the sequences of oligonucleotides CL100 and CL99 represented a region where the pTOMS sequence is only 54% homologous to the sequence of clone F:
• Synthetic oligonucleotides were designed for use as primers for polymerase chain reaction (PCR) amplification of a specific fragment containing the 3' region of the pTOM5 gene with BamHI restriction sites at each end.
• the oligonucleotides (designated 5GENE-5 and 5GENE-3) contain sequences from base 3405 to 3442 of SEQ ID:1 and 1604 to 1630 of the pTOM ⁇ cDNA.
• oligonucleotides were designed for use as primers for polymerase chain reaction (PCR) amplification of a specific fragment containing the phytoene synthase gene promoter with restriction sites at each end (5'- Hindlll : 3'- BamHI).
• the oligonucleotides (designated 5PRO-5 and 5PRO-3) contain sequences from base 1 to 30 and 1155 to 1105 of the phytoene synthase gene:
• the 1151 bp Hindlll/BamHI phytoene synthase gene promoter fragment from the Ml3mpl8 clone (p5PRO) is excised from replicative form DNA and cloned into Hindlll and BamHI cut pTAKl (described in EP 271988 A). Plasmids with the correct orientation of the PS gene promoter are identified by restriction analysis and DNA sequencing. One such clone is designated p5TAK (Fig 4). EXAMPLE 2
• the vector p5TAK (from Example 1.5) is transferred to Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and is used to transform tomato plants. Transformation of tomato stem segments follows standard protocols (eg. Bird et al Plant Molecular Biology 1_1, 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.
• the ripening-specific expression of the ⁇ -glucuronidase (GUS) gene as determined by the phytoene synthase gene promoter is demonstrated by analysis of mature green, breaker and ripening fruit for GUS enzyme activity.
• the response of the gene to exogenous ethylene is demonstrated by incubation of breaker stage fruit in an atmosphere containing additional ethylene followed by analysis of GUS enzyme activity.

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• 1991
  • 1991-03-14 GB GB919105420A patent/GB9105420D0/en active Pending
• 1992
  • 1992-03-12 AU AU13690/92A patent/AU1369092A/en not_active Abandoned
  • 1992-03-12 JP JP4505831A patent/JPH06505871A/ja active Pending
  • 1992-03-12 CA CA 2106091 patent/CA2106091A1/en not_active Abandoned
  • 1992-03-12 EP EP92906497A patent/EP0619844A1/de not_active Withdrawn
  • 1992-03-12 BR BR9205770A patent/BR9205770A/pt not_active Application Discontinuation
  • 1992-03-12 WO PCT/GB1992/000442 patent/WO1992016635A1/en not_active Application Discontinuation

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