Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1993-07-08
1995-08-15
Fox, David T.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 245, 435 691, 435 701, 4351723, 4352404, 4353201, 800205, 800250, C07H 2104, C12N 514, C12N 1582, A01H 500
Patent
active
054420520
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the expression of genes in transgenic plants. In particular it describes the isolation and use of DNA sequences which permit a high level of expression of foreign genes in transgenic plants.
BACKGROUND OF THE INVENTION
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, applied to problems such as the expression of genes in genetically manipulated crop plants. A large number of examples are now 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 500-800 bases in most instances are sufficient to allow for the regulated expression of foreign genes. This regulation has involved tissue-specificity; regulation by external factors such as light, heat treatment, chemicals and hormones; and developmental regulation.
These experiments have been carried out using gene fusions between the promoter sequences and foreign genes such as bacterial promoter genes, etc.
Although regulation has been observed this has been hampered by two factors:
1. The low level of expression observed for the transgene in comparison with the endogenous gene. In most instances expression of the transgene has been approximately 1-10% of the expression achieved when the same promoter drives the endogenous gene. This has led to the suggestion that sequences internal to genes may also be important for efficient expression. This has been supported by experiments in which complete genes including 5' and 3' regions as well as coding regions have been used in blot transformation experiments. The influence of sequences surrounding the introduced transgene on the level of expression which can be achieved is normally referred to as `position effect`. For practical purposes it is desirable that gene constructs introduced into plants give expression levels comparable with that of an endogenous gene. In practice, promoters may be chosen for gene constructs because of their induction pattern, e.g. their tissue specificity or temporal pattern of expression. However, the level of expression of the transgene is usually critical; if the desired promoter cannot give a high enough level of expression it will not be useful.
2. Great variation exists in the level of expression of transgenes between different transformed plant lines. It is not clear why this should be so: it may be another manifestation of the "position effect". These expression levels can differ by as much as two orders of magnitude. Thus a large number of transformants may need to be analysed before one exhibiting the desired expression level can be identified.
These two factors make it very costly and time-consuming to use known promoter constructs for practical genetic plant engineering.
SUMMARY OF THE INVENTION
The object of the present invention is to provide novel plant gene constructs which when used to transform plant cells give a high and reliable expression of the inserted gene.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention we provide a DNA construct for use in transforming plant cells which comprises an exogenous gene under the control of upstream promoter and downstream terminator sequences, characterised in that the upstream promoter is a DNA sequence of not less than about 5 kilobases that is homologous to the DNA control sequence found upstream of the tomato polygalacturonase gene. Preferably that the downstream terminator is a DNA sequence that is homologous to the DNA control sequence of about 1.6 kilobases found downstream of the tomato polygalacturonase g
REFERENCES:
Bird et al., "The tomato polygalacturonase gene and ripening--specific expression in transgenic plants" Plant Molecular Biology, vol. 11, 1988, pp. 651-662--see the whole document.
Shabbeer et al., "Putatuve regulatory factors binding a fruit ripening promoter", Biological Abstracts BR39;65917 see the abstract, vol. 41, 1990 P5-6.
Cordes et al. 1989, Plant Cell 1(10):1025-1034.
Thornburg et al. 1987 Proc. Natl. Acad. Sci. USA 84(3):744-748.
Lewin, R. 1987, Science 237:1570.
Reeck et al. 1987 Cell 50:667.
Bird Colin R.
Grierson Donald
Schuch Wolfgang W.
Fox David T.
Zeneca Limited
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