DNA, DNA constructs, cells and plants derived therefrom

Details DNA, DNA constructs, cells and plants derived therefrom DNA, DNA constructs, cells and plants derived therefrom

1994-09-30

1997-08-19

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...

800255, 800DIG44, 536 232, 4353201, 435411, 435419, A01H 400, C12N 1582, C12N 514, C12N 1529

Patent

active

056591217

DESCRIPTION:

BRIEF SUMMARY
This invention relates to novel DNA constructs, plant cells containing the constructs and plants derived therefrom. In particular it involves the use of antisense or sense RNA technology to control gene expression in plants.
During tomato fruit ripening, the components of the cell walls undergo a series of modifications. The pectin component is subject so substantial modification and degradation. The major changes to the pectin include: solubilisation, depolymerisation and demethylation. The changes in texture of the fruit that occur during ripening occur in parallel with the changes to the pectin component. It has been demonstrated that many of these changes are the result of the action of enzymes in the cell wall.
The cell wall enzymes polygalacturonase and pectin esterase have a major role in modifying pectin during fruit ripening. Individual cDNA clones that corresponded to mRNAs encoding these two enzymes have previously been identified and characterised. DNA sequences from these clones have been used to demonstrate sense and antisense inhibition of these enzymes (EPA 271,988, ICI). For antisense inhibition of, say, pectin esterase, a fragment of DNA coding for the mRNA that produces pectinesterase is inserted into the plant genome under control of a plant promoter, but in reverse orientation. In this way the plant produces RNA sequences complementary to the mRNA that is translated into pectin esterase: and this interferes in some way with the translation of the mRNA into protein. For sense inhibition of, say, polygalacturonase, a fragment of DNA coding for the mRNA that produces polygalacturonase is inserted into the plant genome under control of a plant promoter, in normal orientation. The plant produces RNA sequences homologous with part of the full-length mRNA that is translated into polygalacturonase. Again, this interferes with and can substantially inhibit the translation of polygalacturonase mRNA into protein. How this happens is not clear.
In this invention, we provide clones comprising further novel DNA coding sequences from a new pectin esterase gene. These can be used to make DNA constructs that will control expression of pectinesterase, either by sense or antisense inhibition.
In work leading to the invention, we have identified a novel cDNA (pB8) which encodes a tomato pectin esterase isoenzyme. The cDNA was identified in a clone from a library constructed using mRNA isolated from tomatoes at an early ripening stage. The cDNA in the clone was approximately 2 kb long. Characterisation of the sequence of the clone indicated in addition to sequences similar to those found in the previously characterised cDNA sequence (pPE1), there was also a 358 bp extension at the 5' end. This extended sequence was not predicted since the size of the previous cDNA (pPE1-1.655 kb) corresponded to the determined size of the mRNA (1.6 kb). More recent, unpublished determinations indicate that the true size of the PE mRNA in tomato fruit is approximately 2.0 kb (and see Handa, 1992).
According to the present invention, we further provide recombinant DNA comprising an upstream promoter base sequence, a base sequence for transcription into mRNA under control of said upstream promoter base sequence, and a downstream transcription terminator base sequence, characterised in that the base sequence for transcription comprises a sequence of bases complementary to a substantial run of bases in the novel PE cDNA sequence. The sequence for transcription may be oriented in the normal or reverse sense, so as to produce either sense or antisense RNA in plants. The invention also includes plant cells containing such constructs; plants derived therefrom showing modified ripening characteristics; seeds of such plants; and novel recombinant DNA from clones B8 and B16.
DNA constructs according to the invention comprise a base sequence for transcription at least 10 bases in length. For antisense transcription at least, there is no theoretical upper limit to the base sequence--it may be as long as the relevant mRNA produced by the

REFERENCES:
patent: 5073676 (1991-12-01), Bridges et al.
patent: 5447867 (1995-09-01), Bridges et al.
Harriman et al. (May 1990) Plant Physiol. vol. 93, No. 1, p. 44, abstract 249.
Harriman et al. (1991) Plant Physiology vol. 97 (1) pp. 80-87.
Boswell et al in COMPUTATIONAL MOLECULAR BIOLOGY SOURCES AND METHODS FOR SEQUENCE ANALYSIS (Lesk, ed.) Oxford University Press, Oxford, 1988.
WO,A,9 108 299 (ICI) 13 Jun. 1991, see claims 15; examples 17-24.
Harriman, et al: "Identification and characterization of three pectin methylesterase genes in tomato" PLANT PHYSIOLOGY, vo. 93, No. 1, May 1990, p. 44 -see abstract 249.
Harriman: "Molecular clining and expression of pectin methylesterase, genes in Lycopersicon esculentum (tomato)" CHEMICAL ABSTRACTS, vol. 114, 1991, abstract No. 96207. & PHD THESIS, PURDUE UNIVERSITY, vol. 51, No. 6, 1990, p. 2702.
Harriman et al:, "Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits", PLANT PHYSIOLOGY (1991) vol. 97, No. 1, pp. 80-87, see FIG. 2A.
Tieman, et al: "Inhibition of pectin methylesterase (PME) gene expressioin in transgenic tomato fruit by anti-sense RNA", J. CELL.BIOCHEM.SUPPL.KEYSTONE SYMPOSIUM, vol. 15A, 1991, p. 81, see abstract A353.

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