Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Patent
1992-03-16
1994-11-15
Chereskin, Che S.
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
4351723, 4352404, 4353201, 435250, 435DIG44, 935 67, A01H 400, C12N 514, C12N 1582
Patent
active
053650158
DESCRIPTION:
BRIEF SUMMARY
This application relates to novel DNA constructs, plant cells containing them and plants derived therefrom. In particular it involves the use of antisense RNA technology to control gene expression in plants.
As is well known, a cell manufactures protein by transcribing the DNA of the gene for that protein to produce messenger RNA (mRNA), which is then processed (e.g. by the removal of introns) and finally translated by ribosomes into protein. Translation may be inhibited by the presence in the cell of "antisense RNA". By this term is meant an RNA sequence which is complementary to a sequence of bases in the mRNA in question: complementary in the sense that each base 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. It is believed that this inhibition takes place by formation of a complex between the two complementary strands of RNA, preventing the formation of protein. How this works is uncertain: the complex may interfere with further transcription, processing, transport or translation, or degrade the mRNA, or have more than one of these effects. Such antisense RNA may be produced in a cell by transformation with an appropriate DNA construct arranged to transcribe backwards part of the coding strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).
The use of this technology to downregulate the expression of specific plant genes has been described, in for example European Patent publication no 271988 to ICI (corresponding to U.S. Ser. No. 119,614). Reduction in gene expression has led to a change in the phenotype of the plant: either at the level of gross visible phenotypic difference e.g. lack of anthocyanin production in flower petals of petunia leading to colourless instead of coloured petals (van der Krol et al, Nature, 333, 866-869,1988); or at a more subtle biochemical level e.g. change in the amount of polygalacturonase and reduction in depolymerisation of pectins during tomato fruit ripening (Smith et al, Nature, 334, 724-726, 1988; Smith et al., Plant Mol. Biol., 14, 1990, 369-379). Thus antisense RNA has been proven to be useful in achieving downregulation of gene expression in plants.
The present invention is based on two discoveries: that a known gene of hitherto unknown function is involved in the ethylene biosynthesis pathway; and that RNA antisense to this gene is effective in disrupting this pathway and reducing ethylene production. The gene in question is encoded (almost completely) in pTOM13, disclosed in Holdsworth et al., Nucleic Acids Research, 15, 731-739, 1987.
According to the present invention we provide DNA constructs comprising a DNA sequence homologous to some or all of a gene encoding an enzyme involved in ethylene biosynthesis, preceded by a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.
In a further aspect the invention comprises DNA constructs comprising a transcriptional initiation region operative in plants positioned for transcription of a DNA sequence encoding RNA complementary to a substantial run of bases showing substantial homology to an mRNA encoding an enzyme involved in ethylene biosynthesis. Preferably the mRNA encoding the enzyme involved in ethylene biosynthesis derives from DNA from pTOM13; or from DNA which is fully or partly homologous thereto. The invention also includes plant cells comprising constructs according to the invention; plants derived therefrom showing reduced ethylene production; and seeds of such plants.
Two principal enzymes involved in ethylene biosynthesis in plants are ACC Synthase and ACC Oxidase. We believe that pTOM13 contains DNA coding for the latter. Whether this is so or not, pTOM13 is certainly an effective source of DNA for use in inhibiting ethylene production according to the present invention. See the paper in "Nature", 346 Jul. 19, 1990, Pages 284-287, of which the present
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Grierson Donald
Hamilton Andrew J.
Lycett Grantley W.
Chereskin Che S.
Imperial Chemical Industries plc
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