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
1995-02-28
1997-05-27
Fox, David T.
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...
536 231, 536 241, 435 691, 4351723, 4353201, 435419, 435414, A01H 500, C12N 1511, C12N 1582
Patent
active
056334397
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to the regulation of gene expression in transgenic plants. In particular it is concerned with the isolation and use of DNA sequences which control the expression of genes in lignifying tissues and in response to pathogen attack.
The ability to isolate and manipulate plant genes has opened the way to gain understanding about the mechanisms involved in the regulation of plant gene expression. This knowledge is important for the exploitation of genetic engineering techniques to practical problems such as the expression of genes in genetically manipulated crop plants exhibiting improved quality and production characteristics.
Many examples have been reported in the literature of plant DNA sequences which have been used to drive the expression of foreign genes in plants. In most instances the regions immediately 5' to the coding regions of genes have been used in gene constructs. These regions are referred to as promoter sequences. They may be derived from plant DNA; or from other sources, e.g., viruses. It has been demonstrated that sequences up to 500-1000 bases in most instances are sufficient to allow for the regulated expression of foreign genes. The provide a promoter sequence suitable for the control of foreign gene expression in plants.
SUMMARY OF THE INVENTION
According to the present invention there is provided 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 is or has functional homology to a promoter of a gene of the lignin biosynthesis pathway.
Thus, the present invention comprises the use of the promoter(s) of the cinnamyl alcohol dehydrogenase or other genes involved in the lignin biosynthesis pathway to control the expression of novel and exogenous proteins and genes in these tissues.
Preferably, the promoter is the CAD promoter.
The clone gNtCAD9-6.3SH contains the promoter fragment of this gene. This clone has been deposited at the National Collections of Industrial and Marine Bacteria (NCIB), at 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, on 2nd Apr. 1992 under the reference NCIB Number 40499.
We further provide novel plant cells, and plants transformed with constructs according to the present invention. We illustrate the invention hereinafter using tobacco as a model plant species.
The constructs can be used in the expression of exogenous genes and proteins in vascular tissues, particularly but not exclusively, such as poplar, eucalyptus, pine, and other woody plants as well as forages such as festuca, alfalfa, maize, sorghum, penesitum, amongst others.
Not only will this promoter be expressed in types of regulation exemplified include tissue-specificity, regulation by external factors such as light, heat treatment, chemicals, hormones, and developmental regulation. However, it has also been shown that sequences much longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants.
These experiments have largely been carried out using gene fusions between the promoter sequences and foreign structural genes such as bacterial genes, etc. This has led to the identification of useful promoter sequences.
In the work leading to the present invention we have identified a gene which expresses an enzyme involved in biosynthesis of lignin in vascular plants. We have shown that this gene encodes cimmamyl alcohol dehydrogenase (CAD) which is one of the enzymes specific to the lignin branch pathway of phenylpropanoid metabolism. The gene in question is encoded by the cDNA clone pTCAD19 among others which are the subject of our published copending International Patent Application Number WO 93/05159.
The enzyme encoded by the CAD gene catalyses the oxidation of cinnamyl aldehydes to cinnamyl alcohols which are the direct precursors of the lignin polymer. We have isolated the gene encoding this enzyme from tobacco.
We have shown that C
REFERENCES:
Leyva et al: "cis-Element combinations determine phenylalanine ammonia-lyase gene tissue-specific expression patterns", The Plant Cell, vol. 4, No. 3, Mar. 1992, see whole document.
O'Malley, et al: "Purification, characterization, and cloning of cinnamyl alcohol dehydrogenase in Loblolly Pine (Pinus taeda L.)", Plant Physiology, nol 98, No. 4, Apr. 1992, pp. 1364-1371, see the whole document.
Halpin, et al: "Purification and characterization of cinnamyl alcohol dhydrogenase from tobacco stems", Plant Physiology, vol. 98, No. 1, Jan. 1992, see the whole document.
Kim et al (1994) Plant Mol. Biol 24:105-117.
Fox David T.
McElwain Elizabeth F.
Zeneca Limited
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